Provisional Translation from Japanese Original

8. Compositional standards of each feed additives and standards of manufacturingmethods, etc.
(1) Sodium Alginate
A. Raw material for manufacturing(a) Compositional standards
Physical and chemical properties: This product is white to yellowish-white powder withlittle odor.
Confirmation test:
i. 0.5 g (0.45~0.54 g) of the product is gradually added to 50 mL of water while stirring,
warmed at 60~70 °C for 20 minutes while occasionally stirring to make ahomogeneous solution, and allowed to cool. This is used as a sample solution. When 5
mL of this sample solution is added with 1 mL of calcium chloride test solution,
gelatinous precipitation is generated within 30 seconds.
ii. When 10 mL of the sample solution of i. is added with 1 mL of dilute sulfuric acid,gelatinous precipitation is generated within 30 seconds.
iii. When 1 mL of the sample solution of i. is added with 2 mL of phloroglucinolhydrochloric acid test solution and boiled for 30 seconds, the resulting solution isreddish violet.
iv. The residue obtained by the ashing of this product gives the qualitative reaction of
sodium salt.
Purity test:
i. pH: The pH of the solution (1 → 100) of this product shall be 6.0~8.0.
ii. Sulfate: 0.1 g (0.05~1.4 g) of this product is added with 20 mL of water to make it apaste, shaken well with 1 mL of hydrochloric acid, warmed in a water bath for several
minutes, allowed to cool and filtered. The container is washed with 10 mL of waterand the washings are also filtered with the same filter. Next, the same procedure isrepeated twice with 10 mL of water each. All the filtrates are collected and added withwater to make 50 mL to prepare a sample solution. A control solution is preparedusing 0.4 mL of 0.005 mol/L sulfuric acid by the sulfate test method. When 10 mL of
the sample and control solution are each tested for sulfate, the turbidity of the sample
solution shall not be higher than that of the control solution (0.96 % or less).
iii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is gently heated, carbonized, ashed at450~500 °C, added with 2 mL of hydrochloric acid, and evaporated to dryness on awater bath. The residue is dissolved in 4 mL of diluted acetic acid and 20 mL of waterand filtered. The residue on the filter paper is washed three times with 5 mL of water222

Provisional Translation from Japanese Original

each. All the filtrates are collected and added with water to make 50 mL. This is used
as a sample solution. A control solution is prepared using 2.0 mL of lead standard
solution. When 25 mL each of the sample and control solutions are tested for heavy
metal, the color of the sample solution shall not be darker than that of the control
solution (20 mg/kg or less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
v. Starch: When 5 mL of a solution of this product in water (1 → 100) is added with 2
drops of iodine test solution and shaken, the color of the resulting solution shall not be
blue to reddish violet.
vi. Gelatin: When 5 mL of a solution of this product in water (1 → 100) is added with 1
mL of ammonium molybdate solution (1 → 20) and shaken, precipitation shall not be
generated within 5 minutes.
Loss on drying: 15 % or less (1 g, 105 °C, 4 hours)
Ignition residue: 33~37 % (1 g)(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium alginate areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium alginate
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of sodium
alginate and fillers.
Confirmation test: 1.0 g (0.95~1.04 g) of this product is gradually added to 50 mL of water
while stirring and allowed to stand for an hour while occasionally stirring. Then, thesupernatant obtained by 10 minutes centrifugation at 3,000 rpm is used as a sample
solution, and the confirmation test of the raw materials for manufacturing of sodium
alginate is hereinafter applied mutatis mutandis.
(b) Standard of storage method

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Provisional Translation from Japanese Original

It shall be stored in a capped container.

(2) Ethoxyquin
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 97.5 % or over of ethoxyquin(C14H19NO).
Physical and chemical properties:
i. This product is a yellow-brown to brown viscous fluid with a slight, specific odor.
ii. This product is easy to extremely dissolve in acetone, isopropanol, ethanol, ether,chloroform, petroleum ether or n-hexane and hardly dissolves in water.
iii. This product is gradually colored by air or light.
Confirmation test:
i. When a solution of this product in n-hexane (1 → 10,000) is irradiated with ultraviolet
light (dominant wavelength: 365 nm), it exhibits blue-white fluorescence.
ii. In the measurement of the absorption spectrum of this product in isopropanol solution(1 → 20,000), the absorption maximum is at the wavelengths of 356~362 nm.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of n-hexane. The resulting solution shall be clear and its color shall not be darker
than that of a mixture of 10 mL of a color control stock solution of cobalt chloride, 5
mL of a color control stock solution of copper sulfate, and 5 mL of a color control
stock solution of ferric chloride.
ii. Sulfate: 2.0 g (1.95~2.04 g) of this product is weighed, added with 30 mL of water,
shaken for 2 minutes, and filtered. 15 mL of the filtrate is measured and added with 1
mL of dilute hydrochloric acid and water to make 50 mL. This is used as a sample
solution. When sulfate is tested using a control solution prepared with 0.25 mL of
0.005 mol/L sulfuric acid by the sulfate test method, the turbidity of the sample
solution shall not be higher than that of the control solution (0.012 % or less).
iii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (5 mg/kg or less).

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Provisional Translation from Japanese Original

iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution
by Method No. 3 of the arsenic test method. The arsenic test is performed by themethod using device A. The color of absorbing solution shall not be darker than thestandard color (2 mg/kg or less).
Ignition residue: 0.2 % or less (1 g)Assay: Approximately 0.1 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 1 mL of acetic anhydride and 50 mL of glacial acetic acidfor nonaqueous titration and titrated with 0.1 mol/L of perchloric acid-dioxane solution(potentiometric titration). A blank test is performed in the same way and corrections are
made.
0.1 mol/L of perchloric acid-dioxane solution 1 mL =21.73 mg C14H19NO(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of ethoxyquin are
applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ethoxyquin isapplied mutatis mutandis.
C. Preparation (Part 2: liquid)(a) Compositional standards
This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of ethoxyquin and glycerin, hydrogenated oils, higher saturated fatty acids,
fatty acids, vegetable oil or animal fats.
Content: When this product is determined, it contains ethoxyquin (C14H19NO)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, 0.1 g of the raw material for
manufacturing of ethoxyquin is weighed, dissolved in a little isopropanol, added withn-hexane to make 100 mL and shaken well. The resulting solution is used as a sample
solution. 10 mL of this solution is measured and added with n-hexane to make 100
mL. Using the resulting solution, the confirmation test i. for the raw material for
manufacturing of ethoxyquin is hereinafter applied mutatis mutandis.
ii. 50 mL of the sample solution of i. is measured, the solvent is distilled off under
reduced pressure and the residue is dissolved in 1,000 mL of isopropanol. Using this225

Provisional Translation from Japanese Original

solution, the confirmation test ii. for the raw material for manufacturing of ethoxyquinis hereinafter applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.1 g of ethoxyquin(C14H19NO) is weighed to three significant digits and the value is recorded. It is dissolvedin a little isopropanol, transferred to a 1,000 mL volumetric flask, added with n-hexane,
shaken up well, and added with n-hexane to the graduation line to make 1,000 mL. 1 mLof this solution is measured using a volumetric pipette, transferred to a 100 mL
volumetric flask, and added with n-hexane to the graduation line to make 100 mL. This isused as a sample solution. Separately, approximately 0.1 g of ethoxyquin for assay isweighed to the digits of 0.001 g and the value is recorded. It is dissolved with n-hexane,
transferred to a 1,000 mL volumetric flask, added with more n-hexane to the graduationline to make 1,000 mL, and subjected to the same procedure as that for the sample
solution to prepare a standard solution. For the sample solution (T), the standard solution(S), and n-hexane (B), fluorescence intensities, FT, FS, and FB are measured at theexcitation wavelength 365 nm and the fluorescence wavelength 415 nm using afluorophotometer.
Amount of ethoxyquin (C14H19NO) (mg)=Amount of ethoxyquin for assay (mg)×

FT -FBFS -FB(b) Standard of storage method
It shall be stored in a lightproof airtight container.
D. Preparation (Part 3: powder)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof ethoxyquin and fillers.
Content: When this product is determined,it contains ethoxyquin (C14H19NO) corresponding
to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.1 g of
the raw material for manufacturing of ethoxyquin is weighed, added with 100 mL of nhexane, shaken well and mixed, and filtered to prepare a sample solution. 10 mL ofthis solution is measured and added with n-hexane to make 100 mL. Using thissolution, the confirmation test i. for the raw material for manufacturing of ethoxyquinis hereinafter applied mutatis mutandis.
ii. 50 mL of the sample solution of i. is measured, the solvent is distilled off under
reduced pressure and the residue is dissolved in 1,000 mL of isopropanol. Using this226

Provisional Translation from Japanese Original

solution, the confirmation test ii. for the raw material for manufacturing of ethoxyquinis hereinafter applied mutatis mutandis.
Assay: Methods No. 1 is used for the amount on the label is 40 % and above and Method
No. 2 is used for the label is less than 40 %.
Method No. 1: The amount of this product containing approximately 0.1 g of ethoxyquin(C14H19NO) is weighed to three significant digits and the value is recorded. It is addedwith 50 mL of acetone, shaken up well, and filtered. The residue is washed three times
with 30 mL each of acetone. The filtrate and washings are collected together and thesolvent is distilled off under reduced pressure and nitrogen stream at 40 °C. Within 30
seconds, the residue is dissolved with 1 mL of acetic anhydride and 50 mL of glacialacetic acid for nonaqueous titration, and the assay for the raw material for
manufacturing of ethoxyquin is hereinafter applied mutatis mutandis.
0.1 mol/L perchloric acid-dioxane solution 1 mL =21.73 mg C14H19NO
Method No. 2: The amount of this product containing approximately 0.1 g of ethoxyquin(C14H19NO) is weighed to three significant digits and the value is recorded. It isdissolved with n-hexane, transferred to a 1,000 mL volumetric flask, shaken up well,added with more n-hexane to the graduation line to make 1,000 mL. This solution isfiltered with a dried filter paper and 20 mL of the first filtrate is removed. 1 mL of thesecond filtrate is measured using a volumetric pipette and the assay for ethoxyquin
preparation (part 2: liquid) is hereinafter applied mutatis mutandis.
F -F

Amount of ethoxyquin (C14H19NO) (mg)=Amount of ethoxyquin for assay (mg)× T B
FS -FB(b) Standard of storage method
It shall be stored in a lightproof capped container.

(3) Sodium Caseinate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 3 hours drying at 100 °C, it contains
14.5~15.8 % of nitrogen (N).
Physical and chemical properties: This product is white to pale yellow particles, powder orpieces with no odor or a slight, specific odor.

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Provisional Translation from Japanese OriginalConfirmation test:
i. When 0.1 g (0.05~0.14 g) of this product is dissolved with 10 mL of 1 mol/L sodiumhydroxide test solution, and added with acetic acid to make it slightly acidic, whiteflocculation is produced.
ii. When 0.1 g (0.05~0.14 g) of this product is dissolved with 10 mL of 1 mol/L sodiumhydroxide test solution, added with a drop of copper sulfate test solution and shaken
up, blue precipitation is produced and the solution is purple.
iii. When 0.1 g (0.05~0.14 g) of this product is ignited, it generates smoke and emits a
specific odor. After the smoke generation stop, the heating is stopped and the productis allowed to cool. The black residue is added with 5 mL of dilute nitric acid, warmed
and filtered. When the filtrate is added with 1 mL of ammonium molybdate test
solution and warmed, yellow precipitation is produced.
iv. The residue on ignition of this product gives the flame reaction of sodium salt.
Purity test:
i. Clarity and color of solution: This product is dried in a desiccator (reduced pressure,sulfuric acid) for 4 hours to make it fine powder. 0.1 g (0.05~0.14 g) of this powder isweighed, added with 30 mL of water, shaken up, allowed to stand for approximately
10 minutes, added with 2 mL of 0.1 mol/L sodium hydroxide test solution, andwarmed at 60 °C for one hour with occasional shaking to make it dissolved. When it isallowed to cool and then added with water to make 100 mL, it is colorless and its
turbidity shall be slightly cloudiness or clearer.
ii. pH: The pH of the solution (1 → 50) of this product shall be 6.0~7.5.
iii. Fat: Approximately 2.5 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is added with 15 mL of hydrochloric acid (27 → 40), dissolved bygentle direct heating, and then heated in a water bath for 20 minutes. It is allowed tocool, added with 10 mL of ethanol, transferred to a rohrig tube, added with 25 mL ofether, and vigorously shaken up for 1 minute. Then, the mixture is added with 25 mLof petroleum ether, vigorously shaken up for 30 seconds, and allowed to stand. The
upper layer solution obtained from the branch tube (A) is filtered, and the filtrate isplaced in a flask, whose mass is known. Then, it is extracted twice with 15 mL of ether
and 15 mL of petroleum ether in the same way. The upper layer solutions are collectedin the previous flask, and ether and petroleum ether are distilled off on a water bath.
When the residue is dried at 98~100 °C for 4 hours, the amount of residue shall be
1.5 % or less.

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Provisional Translation from Japanese OriginalRohrig tube
(Unit: mm)
iv. Heavy metal: 2.0 g (1.95~2.04 g) of this product is gently heated and carbonized.
Then it is ashed at less than 500 °C, added with 2 mL of hydrochloric acid, andevaporated to dryness on a water bath. The residue is resolved in 4 mL of diluted
acetic acid and 20 mL of water and filtered. Then, the residue on the filter paper iswashed three times with 5 mL of water each. These filtrates are collected together andadded with 50 mL of water. The resulting solution is used as a sample solution. A
control solution is prepared using 2.0 mL of lead standard solution. When heavy metal
is tested using 25 mL of the sample and control solutions each, the color of the sample
solution shall not be darker than that of the control solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution by
Method No. 3 of the arsenic test method. The arsenic test is performed by the method
using device A. The color of absorbing solution shall not be darker than the standard
color (2 mg/kg or less).
Loss on drying: 15 % or less (1 g, 100 °C, 3 hours)
Ignition residue: 6 % or less (1 g)Assay: This product is dried at 100 °C for 3 hours, approximately 0.15 g of it is weighed tothe digits of 0.001 g and the value is recorded. The assay is performed by the nitrogen
determination method (Kjeldahl method).
0.05 mol/L sulfuric acid 1 mL = 1.401 mg N(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium caseinate
are applied mutatis mutandis.
(b) Standard of storage method229

Provisional Translation from Japanese Original

The standard of storage method of the raw material for manufacturing of sodium
caseinate is applied mutatis mutandis.

(4) Sodium Carboxymethyl Cellulose
A. Raw material for manufacturing(a) Compositional standards
Physical and chemical properties: This product is white to whitish powder, particles, or
fibrous substances with no odor.
Confirmation test:
i. 0.5 g (0.45~0.54 g) of this product is gradually added to 50 mL of water while stirring
and warmed at 60~70 °C for 20 minutes while occasionally stirring, then allowed tocool. This is used as a sample solution. The volume required for testing is measured
and diluted fivefold with water. When a drop of this diluted solution is added with 0.5
mL of chromotropic acid test solution and heated in a water bath for 10 minutes, itturns red-purple.
ii. When 5 mL of the sample solution of i. is shaken up with 10 mL of acetone, whiteflocculation is produced.
iii. When 5 mL of the sample solution of i. is shaken up with 1 mL of copper sulfate test
solution, pale blue flocculation is produced.
iv. The residue obtained by the ashing of this product gives the qualitative reaction of
sodium salt.
Purity test:
i. pH: The pH of the solution (1 → 100) of this product shall be 6.0~8.5.
ii. Chloride: 0.1 g (0.05~0.14 g) of this product is added with 20 mL of water and 0.5 mLof hydrogen peroxide solution and heated in a water bath for 30 minutes. Aftercooling, it is added with water to make 100 mL and filtered through filter paper. 25
mL of this filtrate is measured and added with 6 mL of dilute nitric acid to prepare asample solution. When chloride is tested, using a control solution prepared with 0.45
mL of 0.01 mol/L hydrochloric acid by the chloride test method, the turbidity of thesample solution shall not be higher than that of the control solution (0.64 % or less).
iii. Sulfate: 20 mL of the filtrate obtained in ii. is measured and added with 1 mL of
dilute hydrochloric acid and water to make 50 mL. This solution is used as a sample
solution. When sulfate is tested, using a control solution prepared with 0.4 mL of 0.05
mol/L sulfuric acid by the sulfate test method, the turbidity of the sample solution shallnot be higher than that of the control solution (0.96 % or less).

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Provisional Translation from Japanese Originaliv Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution by
Method No. 3 of the arsenic test method. The arsenic test is performed by the method
using device A. The color of absorbing solution shall not be darker than the standard
color (4 mg/kg or less).
Loss on drying: 12 % or less (1 g, 105 °C, 4 hours)(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium
carboxymethyl cellulose are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
carboxymethyl cellulose is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder, particles, or fibrous substances of the mixture of the raw material
for manufacturing of sodium carboxymethyl cellulose and fillers.
Confirmation test: According to the amount of this product on the label, 0.05 g of the raw
materials for manufacturing of sodium carboxymethyl cellulose is weighed, gradually
added to 50 mL of water with stirring, warmed at 60~70 °C for 20 minutes whileoccasionally stirring, and allowed to cool to prepare a sample solution. The confirmation
test for the raw material for manufacturing of sodium carboxymethyl cellulose ishereinafter applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
carboxymethyl cellulose is applied mutatis mutandis.

(5) Formic Acid
A. Raw material for manufacturing

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Provisional Translation from Japanese Original(a) Compositional standards
Content: When this product is determined, it contains 98.0 % or over of formic acid
(CH2O2).
Physical and chemical properties:
i. This product is clear liquid with a specific acid odor.
ii. This product is miscible with water, ethanol, acetone or ether.
Confirmation test:
i. The pH of the solution of this product in water (1 → 100) is 2.0~2.3.
ii. When 1 mL of this product is added with 1 mL of ethanol and 3 drops of sulfuric acidand warmed on a water bath, it emits the odor of ethyl formate.
iii. When this product is added with lead acetate solution, white crystalline precipitationis generated. When it is added with silver nitrate solution and heated, it becomescloudy within 30 seconds.
Purity test:
i. Residue on evaporation: When 50 g (49.5~50.4 g) of this product is evaporated on awater bath and dried at 105~110 °C to constant weight, and the residue shall be 5 mg
or less (0.01 % or less).
ii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution
by Method No. 1 of the arsenic test method. The arsenic test is performed by themethod using device A. The color of absorbing solution shall not be darker than thestandard color (2 mg/kg or less).
iv. Chloride: 20 g (19.5~20.4 g) of this product is weighed to prepare a sample solution
by the chloride test method. A control solution is prepared using 0.3 mL of 0.01 mol/Lhydrochloric acid. When chloride is tested using these samples and control solutions,the turbidity of the sample solution shall not be higher than that of the control solution(5 mg/kg or less).
Assay: Approximately 2.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is dissolved with 100 mL of newly boiled and cooled water and titrated
with 1 mol/L sodium hydroxide solution (indicator: 3 drops of phenolphthalein test
solution). A blank test is performed in the same way and corrections are made.
1 mol/L sodium hydroxide solution 1 mL = 46.03 mg CH2O2

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Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in an acid resistance airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of formic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of formic acid isapplied mutatis mutandis.
(c) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
Precautions: Handle this feed additive with care because of its low pH.
C. Preparation (Part 2: liquid)(a) Compositional standards
This product is liquid, in which the raw material for manufacturing of formic acid ismixed with water and added with food dye if necessary.
Content: When this product is determined, it contains formic acid (CH2O2) corresponding to
97~103 % of the amount on the label.
Confirmation test:
i. The confirmation test i. for the raw material for manufacturing of formic acid is appliedmutatis mutandis.
ii. The confirmation test ii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
iii. The confirmation test iii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
Assay: The amount of this product containing approximately 0.5 g of formic acid (CH2O2)
is weighed to three significant digits and the value is recorded. It is dissolved with 100
mL of newly boiled and cooled water and titrated with 1 mol/L sodium hydroxide
solution (potentiometric titration).
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of formic acid isapplied mutatis mutandis.
(c) Standards of the label
The standards of the label of the preparation (Part 1) of formic acid are applied mutatis
mutandis.

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Provisional Translation from Japanese Original

D. Preparation (Part 3: liquid)(a) Compositional standards
This product is liquid, in which the raw material for manufacturing of formic acid ismixed with water, mixed with ammonia (limited to one meeting the standards of foodadditives) at a molar ratio of ammonia to formic acid, 1:4, and if necessary added with fooddye.
Content: When this product is determined, it contains formic acid (CH2O2) corresponding to
97~103 % of the amount on the label.
Confirmation test:
i. The pH of the solution of this product in water (1 → 100) is 2.8~3.2.
ii. The confirmation test ii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
iii. The confirmation test iii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
Assay: The assay of storage method of the preparation (Part 2) of formic acid is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of formic acid isapplied mutatis mutandis.
(c) Standards of the label
The standards of the label of the preparation (Part 1) of formic acid are applied mutatis
mutandis.
E. Preparation (Part 4)(a) Compositional standards
This product is powder or particles in which the raw material for manufacturing of formic
acid is added with water as appropriate, and then mixed with fillers.
Content: When this product is determined, it contains formic acid (CH2O2) corresponding to
90~110 % of the amount on the label.
Confirmation test:
i. 5 g (4.5~5.4 g) of this product is weighed, added with 100 mL of water, stirred for 1
minute and filtered. The pH of the filtrate is 1.6~2.1.
ii. To 1 mL of the filtrate of i., the confirmation test ii. for the raw material for
manufacturing of formic acid is applied mutatis mutandis.
iii. To the filtrate of i., the confirmation test iii. for the raw material for manufacturing of
formic acid is applied mutatis mutandis.

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Provisional Translation from Japanese Original

Assay: Approximately 2.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is added with 20 mL of newly boiled and cooled water to be suspended,and titrated with 1 mol/L sodium hydroxide solution (indicator: 3 drops ofphenolphthalein test solution). A blank test is performed in the same way and corrections
are made.
1 mol/L sodium hydroxide solution 1 mL = 46.03 mg CH2O2(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of formic acid isapplied mutatis mutandis.
(c) Standards of the label
The standards of the label of the preparation (Part 1) of formic acid are applied mutatis
mutandis.
F. Preparation (Part 5: liquid)(a) Compositional standards
Formic acid formulation is a liquid obtained by mixing sodium hydroxide VS [limited tothose conforming to the standards for food additives (excluding the part pertaining to theclarity and color of solution); however, the term “not more than 2.0%” in the “sodium
carbonate” section shall be deemed to be replaced with “not more than 3.0%”] with anactive ingredient for formic acid production at a molar ratio of 5.4 to 1 (formic acid to
sodium hydroxide equivalent).
Content: When this product is determined, it contains formic acid (CH2O2) corresponding to
97~103 % of the amount on the label.
Confirmation test:
i. The pH of the solution of this product in water (1 → 100) is 2.6~3.2.
ii. The confirmation test ii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
iii. The confirmation test iii. for the raw material for manufacturing of formic acid isapplied mutatis mutandis.
Assay: The assay of storage method of the preparation (Part 2) of formic acid is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of formic acid isapplied mutatis mutandis.
(c) Standards of the label
The standards of the label of the preparation (Part 1) of formic acid are applied mutatis
mutandis.

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Provisional Translation from Japanese Original(6) Glycerin Fatty Acid Ester
A. Raw material for manufacturing(a) Compositional standards
This product is esters of fatty acids and glycerin or polyglycerin and their derivatives.
This product includes glycerin fatty acid ester, glycerin lactic acid fatty acid ester, glycerin
citric acid fatty acid ester, glycerin succinic acid fatty acid ester, glycerin diacetyl tartaricacid fatty acid ester, polyglycerin fatty acid ester, and polyglycerin condensed ricinoleic
acid ester.
Physical and chemical properties: This product is colorless to brown powder, flakes, coarse
powder, particulate or waxy blocks, semiliquid, or liquid, with no odor or a specific odor.
Confirmation test:
i. Approximately 5 g of this product is added with 50 mL of dilute potassium hydroxideethanol test solution and heated with a reflux condenser in a water bath for an hour,then distill ethanol until almost dryness. Then, it is added with 50 mL of hydrochloric
acid (1 → 10) and shaken up well. The produced fatty acids are separated by 3
extractions with 40 mL each of a mixture of petroleum ether and methyl ethyl ketone(7:1). This aqueous layer is stirred well, added with sodium hydroxide solution (1 →9) to make it almost neutral, and concentrated in a water bath under reduced pressure.
It is added with 20 mL of methanol at approximately 40 °C, shaken up well, cooled,
and filtered. Methanol of the filtrate is distilled away in a water bath. A solution of thisresidue in methanol (1 → 10) is used as a sample solution. Separately, 9 mL of
methanol is added with 1 mL of glycerin to prepare a standard solution. 5 µL each of
the sample and standard solutions are spotted on a thin layer plate prepared using silica
gel for thin-layer chromatography. Then, they are developed approximately 15 cm
from the base line with the developing solvent, a mixture of n-butanol, methanol, andchloroform (5:3:2), and then the thin layer plate is air dried and heated at 110 °C for 10
minutes to remove the solvent. When the cooled thin layer plate is sprayed withthymol-sulfuric acid test solution, heated at 110 °C for 20 minutes to give a color,white spots are observed in the same location of the sample solution for glycerin ester,
and white spots or white linear spots are observed in the same location or under the
sample solution for polyglycerin ester.
ii. The petroleum ether and methyl ethyl ketone layers obtained by separation in i. arecollected and the solvent is distilled away, and then oily matter or white to ayellowish-white solid remains. When 0.1 g (0.05~0.14 g) of this residue is shaken with
5 mL of ether, it is dissolved.

236

Provisional Translation from Japanese Originaliii. Except the cases of glycerin fatty acid ester and polyglycerol ester, when 5 mL of thesample solution of i. and 50 mL of water are mixed by shaking, the resulting solution
gives the reaction of lactate for glycerin lactic acid fatty acid ester, the reaction of
citrate ii. for glycerin citric acid fatty acid ester, the reaction of succinate for glycerin
succinic acid fatty acid ester, and the reaction of acetate and tartrate for glycerindiacetyl tartaric acid fatty acid ester.
iv. In the case of polyglycerin condensed ricinoleic acid ester, it is mixed with thepetroleum ether and methyl ethyl ketone layers obtained by separation in i. and thesolution is washed twice with 50 mL each of water, dehydrated with anhydrous
sodium sulfate, filtered, and warmed under reduced pressure to remove the solvent.
Approximately 1 g of the residue is weighed to the digits of 0.01 g, the value isrecorded, and the residue is placed in a round-bottom flask as shown in the figure
below. Then, it is added with 5 mL of acetic anhydride-pyridine test solution using a
volumetric pipette. A small funnel is placed on the mouth of the round-bottom flask
and the flask is heated in an oil bath at 95~100 °C for a drop with the height of 1 cm
from the bottom immersed in the oil. It is allowed to cool and added with 1 mL ofwater, shaken well, further heated for 10 minutes, and allowed to cool. Then, thefunnel and the neck of the flask are washed with 5 mL of ethanol and excessive
volume of acetic acid is titrated with 0.5 mol/L potassium hydroxide-ethanol solution(indicator: 1 mL of phenolphthalein test solution). Separately, a blank test is
performed. The hydroxyl value calculated by the following equation is 150~170.
Provided, however, that the acid value is measured using approximately 0.5 g of theresidue.
Hydroxyl value =(a-b)×28.05+Acid value
Amount of the residue weighed (g)a: Consumed volume of 0.5 mol/L potassium hydroxide-ethanol solution in the blanktest (mL)b: Consumed volume of 0.5 mol/L potassium hydroxide-ethanol solution in the main
test (mL)237

Approx. 200

Approx. 120

Provisional Translation from Japanese Original

Approx. 77(Outer diameter)(Unit mm)
Purity test:i. Acid value: According to the acid value of the sample, the amount of the sample to becollected of the sample, as shown in the following table, is weighed to three significant
digits and the value is recorded. It is added with 50 mL of the mixture of ethanol andether (1:1) and as appropriate warmed to be dissolved to prepare a sample solution. It
is allowed to cool and added with 0.1 mol/L potassium hydroxide-ethanol solution
until the solution continuously gives a red color for 30 seconds using an indicator,
phenolphthalein test solution. The acid value calculated by the following equation
shall be 6.0 or less for glycerin fatty acid ester and glycerin lactic acid fatty acid ester,
12 or less for polyglycerin fatty acid ester and polyglycerin condensed ricinoleic acid
ester, 100 or less for glycerin citric acid fatty acid ester, 60~120 for glycerin succinicacid fatty acid ester and glycerin diacetyl tartaric acid fatty acid ester. However, thesolvent to be used is previously added with 0.1 mol/L potassium hydroxide-ethanol
solution until the solution continuously gives a red color for 30 seconds using an
indicator, phenolphthalein test solution.
Acid value =Consumed volume of 0.1 mol⁄Lpotassium hydroxide-ethanol solution (mL)×5.611
Amount of the residue weighed (g)TableAcid value

Amount of the sample to be collected

Less than 5

10 g

5 to less than 15

5g

238

Provisional Translation from Japanese Original

15 to less than 50

3g

50 to less than 120

1g

120 or over

0.5 g

ii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by Method No. 3 of the arsenic test method. The arsenic test is performed by themethod using device A. The color of absorbing solution shall not be darker than thestandard color (4 mg/kg or less).
iv. Polyoxyethylene: 1.0 g (0.95~1.04 g) of this product is weighed, placed in a 200 mL
flask, added with 25 mL of dilute potassium hydroxide-ethanol test solution. It isboiled with a ground glass joint reflux condenser in a water bath for an hour withoccasional shaking. Then, it is evaporated to dryness by distilling away ethanol in awater bath or under the reduced pressure, added with 20 mL of sulfuric acid (3 →100), shaken up well while warming, added with 15 mL of ammonium thiocyanatecobalt nitrate test solution, shaken up well, added with 10 mL of chloroform, again
shaken up and allowed to stand. The chloroform layer is not blue.
Ignition residue: 1.5 % or less (1 g)(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of glycerin fatty acid
ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of glycerin fatty
acid ester is applied mutatis mutandis.

(7) Dibutylhydroxytoluene
A. Raw material for manufacturing(a) Compositional standards

239

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains 98.5 % or over of
dibuthylhydroxytoluene (C15H24O)
Physical and chemical properties:
i. This product is colorless crystals or white crystalline powder or blocks, with no odor ora slight, specific odor, and with no taste.
ii. This product is easy to extremely dissolve in acetone, easy to dissolve in ethanol, andhardly dissolves in water.
Confirmation test:
i. When 5 mg (4.5~5.4 mg) of this product is added with 1~2 drops of a solution of 5nitroso-8-oxyquinoline in sulfuric acid (1 → 100), it dissolves while becoming yellow,
and then the color of the solution changes to red-brown.
ii. When 1 mL of a solution of this product in ethanol (1 → 30) is added with 3~4 dropsof dilute ferric chloride test solution, the resulting solution gives no color, and it issubsequently added with small crystals of α-α'-dipyridyl, it is red. However, a dilute
ferric chloride test solution which gives no color in a blank test shall be used.
Purity test:
i. Melting point: The melting point of this product shall be 69.5~71.5 °C.
ii. Freezing point: The solidifying point of this product shall be 69.0 °C or over.
iii. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is added with
10 mL of ethanol to be dissolved, the resulting solution shall be colorless and clear.
iv. Sulfate: 0.5 g (0.45~0.54 g) of this product is weighed, added with 30 mL of water,heated in a water bath for 5 minutes with occasional shaking, allowed to cool, filtered,and added with 1 mL of dilute hydrochloric acid and water to make 50 mL. This isused as a sample solution. When sulfate is tested, using a control solution preparedwith 0.2 mL of 0.05 mol/L sulfuric acid by the sulfate test method, the turbidity of thesample solution shall not be higher than that of the control solution (0.019 % or less).
v. Heavy metal: When 0.5 g (0.45~0.54 g) of this product is weighed, dissolved with 35
mL of acetone, added with 2 mL of diluted acetic acid and water to make 50 mL, andadded with 2 drops of sodium sulfide test solution, the color of the resulting solution
shall not be darker than that of a solution in which 35 mL of acetone is added with 2
mL of lead standard solution, 2 mL of diluted acetic acid and water to make 50 mL
and added with 2 drops of sodium sulfide test solution (40 mg/kg or less).
vi. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution
by Method No. 3 of the arsenic test method. For the sample solution, when the arsenic
test is performed by the method using device A, the color of absorbing solution shallnot be darker than the standard color (2 mg/kg or less).

240

Provisional Translation from Japanese Originalvii. p-cresol: 1.0 g (0.95~1.04 g) of this product is weighed, added with 10 mL of waterand 1 mL of strong ammonia solution, heated in a water bath for 3 minutes withoccasional shaking, is allowed to cool and is filtered. The residue on the filter paper iswashed with a little water. The washings and the filtrate are mixed and added withwater to make 100 mL. This solution is used as a sample solution. When 3 mL of thesample solution is measured, transferred to a Nessler tube, added with 1 mL of asolution of phosphomolybdic acid in ethanol (1 → 20) and 0.2 mL of ammonia test
solution, shaken up, added with water to make 50 mL, and allowed to stand for 10
minutes, the color of the resulting solution shall not be darker than that of a solution
prepared with 3 mL of p-cresol solution (1 → 100,000) by the same procedure for the
preparation of the sample solution.
Water content: 0.20 % or less (direct titration)
Ignition Residue: 0.05 % or less (2 g)Assay: The amount required for testing of this product is weighed to three significant digits,
and tested by the freezing point measurement method.
Amount of dibutylhydroxytoluene (C15 H24 O) (%) = 1.6976 T - 18.5822
T: Freezing point(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of
dibutylhydroxytoluene are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of
dibutylhydroxytoluene is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of
dibutylhydroxytoluene and fillers.
Content: When this product is determined, it contains dibuthylhydroxytoluene (C15H24O)
corresponding to 90~110 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of
dibutylhydroxytoluene is applied mutatis mutandis.
Ignition Residue: 2.4 % or less (1 g)

241

Provisional Translation from Japanese Original

Assay: The assay of the raw material for manufacturing of dibutylhydroxytoluene is appliedmutatis mutandis.
Amount of dibutylhydroxytoluene (C15 H24 O) (%) = 1.6976 T - 18.5822 - R
T: Freezing point
R: Ignition Residue (%)(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of
dibutylhydroxytoluene is applied mutatis mutandis.

(8) Sucrose Fatty Acid Ester
A. Raw material for manufacturing(a) Compositional standards
This product is an ester of fatty acids derived from animal fat and plant oil and sucrose.
Physical and chemical properties: This product is white to yellow-brown powder or blocks
or colorless to slightly yellow viscous resinoids with no odor or a slight, specific odor.
Confirmation test:i. 1 g (0.5~1.4 g) of this product is added with 25 mL of potassium hydroxide-ethanol test
solution, and heated with a reflux condenser in a water bath for an hour. This solution
is added with 50 mL of water and distilled until approximately 30 mL of the residue
liquid. After cooling, the residue liquid is added with 10 mL of dilute hydrochloric
acid, shaken up well, saturated with sodium chloride, extracted twice with 30 mL eachof ether. The ether layer is collected, washed with 20 mL of saturated sodium chloride
solution, and dehydrated with 2 g (1.5~2.4 g) of anhydrous sodium sulfate, and ether isdistilled away and then sufficiently removed by blowing air. When the residue iscooled to 10 °C, oil drops or a colorless to pale yellow-brown solid is deposited.
ii. 2 mL of the aqueous layer, which is separated from the ether layer in the test i., ismeasured, transferred to a test tube, warmed in a water bath to remove ether, thenallowed to cool. When 1 mL of anthrone test solution is added slowly along the wall of
the tube to the solution to form a layer, the boundary surface is blue to green.
Purity test:i. Acid value: Approximately 3 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is dissolved in 60 mL of a mixture of isopropanol and water (2:1)
and titrated with 0.1 mol/L of potassium hydroxide solution (indicator: 1 mL ofphenolphthalein test solution). Separately, a blank test is performed in the same way.
The acid value calculated by the following equation shall be 6 or less.

242

Provisional Translation from Japanese Original

Acid value =Consumed volume of 0.1 mol/L of potassium hydroxide solution mL × 5.611
Amount of sample weighed (g)ii. Dimethylformamide: Approximately 10 g of this product is weighed to the digits of
0.1 g and the value is recorded. It is placed in a 200 mL flask, added with 100 mL of atest solution of 5 % sodium hydroxide in methanol. On the flask a reflux condenser
is placed, and then a condenser is connected to the reflux condenser. The head of thecondenser is previously immersed in 10 mL of a solution of hydrochloric acid inmethanol (1 → 100) in a receiver. The flask is heated in a water bath for 30 minutes.
Then, the water in the reflux condenser is removed and solution is distilled to collect
50 mL of distillate. The distillate is concentrated to almost dryness in a water bath andthe residue is dissolved with 10 mL of water and transferred to a separatory funnel.
The receiver is washed three times with 10 mL each of water. The washings are mixed
in the solution in the separatory funnel. This solution is added with 10 mL of a mixtureof carbon disulfide and chloroform (1:20) and 5 mL of aqueous ammonia solution (1→ 3) and vigorously shaken up for 20 minutes. It is added with 1 mL of copper
sulfate-ammonia test solution, vigorously shaken up for 1 minute, added with 5 mL ofacetic acid solution (1 → 3 ) and then vigorously shaken up for 1 minute. The lower
layer is collected and dehydrated with anhydrous sodium sulfate. This solution is used
as a sample solution. The color of this solution shall not be darker than that of asolution prepared in the way in which 10 mL of dimethylamine hydrochloride standard
solution is measured and placed in a separatory funnel, added with 30 mL of water andadded with 10 mL of a mixture of carbon disulfide and chloroform (1:20) andhereinafter the above mentioned procedure is performed (1 mg/kg or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less)iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by Method No. 3 of the arsenic test method. For the sample solution, when the arsenic
test is performed by the method using device A, the color of absorbing solution shallnot be darker than the standard color (4 mg/kg or less).
v. Free sucrose: Approximately 2 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is added with 40 mL of n-butanol and warmed on a water bathto be dissolved and extracted twice with 20 mL each of sodium chloride solution (1 →243

Provisional Translation from Japanese Original20). The extracts are mixed together, added with 2 mL of dilute hydrochloric acid, andheated in a water bath for 30 minutes, then allowed to cool, added with 2~3 drops ofphenolphthalein test solution, neutralized with 1 mol/L sodium hydroxide test solution,
and added with water to make 100 mL. This solution is used as a sample solution. 20
mL of this sample solution is measured, added with 20 mL of Bertrand’s test solution
A and 20 mL of Bertrand’s test solution B, gently boiled for 3 minutes, and allowed tostand to precipitate cuprous oxide. At this time, the supernatant shall be indigo blue.
Then, the supernatant is filtered with a glass filter. The precipitation in the flask is
dissolved with 20 mL of Bertrand’s test solution C, is filtered with the same glass filter
as mentioned before, and washed with water. The mixture of washings and the filtrate
is titrated with Bertrand’s test solution D. When by the following equation the content
of free sugar is determined using the amount of copper calculated from the obtained
titer and the amount of invert sugar specified in the Table of Bertrand’s determinationof sugar, it shall be 10 % or less.
Content of free sugar =Amount of invert sugar (mg) × 0.95 × 5
× 100 (%)
Amount of the sample weighed (mg)Water content: Approximately 500 mg of this product is weighed to the digits of 1 mg andthe value is recorded. The amount of water obtained in the test by the back titration
method of the water determination method (Karl Fischer method) shall be 4 % or less.
However, the test solution is measured, transferred to a dry titration flask, added with 10
mL of methanol for Karl Fischer, added with a certain volume of Karl Fischer test
solution to make it have an excess of approximately 10 mL. The flask is sealed with astopper. The solution is stirred for 20 minutes, and then titrated with water-methanol
standard solution with vigorously stirring. Separately, a blank test is performed in thesame way and corrections are made.
Ignition Residue: 1.5 % or less (1 g)(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sucrose fatty acid
ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sucrose fatty
acid ester is applied mutatis mutandis.

244

Provisional Translation from Japanese Original(9) Sorbitan fFty Acid Ester
A. Raw material for manufacturing(a) Compositional standards
This product is an ester of fatty acids derived from animal fat and plant oil and sorbitan.
Physical and chemical properties: This product is white to yellow-brown liquid, powder,flakes, particles, or waxy blocks.
Confirmation test:
i. When 0.5 g (0.45~0.54 g) of this product is added with 5 mL of absolute ethanol,
heated to be dissolved, added with 5 mL of dilute sulfuric acid, heated in a water bathfor 30 minutes, and cooled, oil drops or a white to yellow-white solid is deposited.
When the oil drops or solid is separated, added with 5 mL of ether and shaken up, itdissolves.
ii. When 2 mL of the remaining solution after the separation of fat and oil or solid in i.is
measured, added with 2 mL of newly prepared catechol solution (1 → 10), shaken up,
and added with 5 mL of sulfuric acid and shaken up, the resulting solution is red to
red-brown.
Purity test:i. Acid value: Approximately 5 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is dissolved with 100 mL of a mixture of ethanol and ether (1:1)
by warming, allowed to cool, added with 1 mL of phenolphthalein test solution, andtitrated with 0.1 mol/L potassium hydroxide-ethanol solution. Separately, a blank test
is performed in the same way. The acid value calculated by the following equation
shall be 15 or less.

Acid value =consumed volume of 0.1 mol/L of potassium hydroxide-ethanol solution (mL)× 5.611Amount of the sample weighed (g)ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less)iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by Method No. 3 of the arsenic test method. For the sample solution, when the arsenic

245

Provisional Translation from Japanese Original

test is performed by the method using device A, the color of absorbing solution shallnot be darker than the standard color (4 mg/kg or less).
Ignition Residue: 1.5 % or less (1 g)(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sorbitan fatty acid
ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sorbitan fatty
acid ester is applied mutatis mutandis.

(10) Butylated hydroxyanisole
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 99.0 % or over of butylatedhydroxyanisole (C11H16O2)
Physical and chemical properties:

i. This product is white or slightly yellow-brown granules, blocks, or powder, with aslight, specific odor and with an acrid taste.
ii. This product is easy to extremely dissolve in ethanol or acetone and hard to extremely
dissolve in water.
Confirmation test:
i. When 2~3 mL of a solution of this product in ethanol (1 → 100) is shaken up with 2~3
drops of sodium borate solution (1 → 50) and small crystals of 2,6dibromquinonechloroimide, the color of the resulting solution is livid.
ii. When 1 mL of a solution of this product in ethanol (1 → 30) is added with 3~4 dropsof dilute ferric chloride test solution, the resulting solution gives no color, and it issubsequently added with small crystals of α-α'-dipyridyl, it is red. However, a dilute
ferric chloride test solution which gives no color in a blank test shall be used.
Purity test:
i. Melting point: The melting point of this product shall be 62~65 °C.
ii. Clarity and color of solution: When 0.5 g (0.45~0.54 g) of this product is dissolved
with 10 mL of ethanol, the solution shall be colorless and clear.

246

Provisional Translation from Japanese Originaliii. Sulfate: 0.5 g (0.45~0.54 g) of this product is weighed, dissolved with 35 mL ofacetone, added with 1 mL of dilute hydrochloric acid and water to make 50 mL. Thisis used as a sample solution. 0.2 mL of 0.005 mol/L sulfuric acid is added with 35 mLof acetone, 1 mL of dilute hydrochloric acid and water to make 50 mL by the sulfate
test method. This is used as a control solution. When sulfate is tested, using the control
solution, the turbidity of the sample solution shall not be higher than that of the control
solution (0.019 % or less).
iv. Heavy metal: When 0.5 g (0.45~0.54 g) of this product is weighed, dissolved with 35
mL of acetone, added with 2 mL of diluted acetic acid and water to make 50 mL. Thisis used as a sample solution. The color of the sample solution shall not be darker thanthat of a solution in which 35 mL of acetone is added with 2 mL of lead standard
solution, 2 mL of diluted acetic acid and water to make 50 mL (40 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution by
Method No. 3 of the arsenic test method. For the sample solution, when the arsenic test
is performed by the method using device A, the color of absorbing solution shall not
be darker than the standard color (2 mg/kg or less).
vi. Para hydroxyanisole: 1.0 g (0.95~1.04 g) of this product is weighed and dissolved
with 20 mL of a mixture of ether and petroleum benzin (1:1). This solution is addedwith 10 mL of water and 1 mL of 1 mol/L sodium hydroxide test solution, shaken up,
and allowed to stand. Of the top and bottom layers, the bottom layer is collected. It is
added with 20 mL of a mixture of ether and petroleum benzin (1:1), shaken up, andallowed to stand, and the bottom layer is collected and added with water to make 500
mL. 1 mL of this solution is transferred to a Nessler tube and added with 2 mL of 1
mol/L sodium hydroxide test solution, 5 mL of boric acid solution (3 → 100) andwater to make 30 mL. When this is added with 5 mL of 4-aminoantipyrine solution (1→ 1,000), shaken up, then added with 1 mL of potassium ferricyanide solution (1 →100), shaken up, added with water to make 50 mL and allowed to stand for 15 minutes,
the color of the resulting solution shall not be darker than that of a solution preparedby which 0.6 mL of the color control stock solution of cobalt chloride is added withwater to make 50 mL (approximately 0.5 % or less).
Water content: 0.30 % or less (direct titration)
Ignition Residue: 0.05 % or less (2 g)Assay: Approximately 0.3 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 50 mL of acetone, added with 50 mL of water and 20 mL
of dilute sulfuric acid, and titrated with 0.1 mol/L ceric ammonium sulfate solution

247

Provisional Translation from Japanese Original(indicator: 0.2 mL of o-phenanthroline test solution). A blank test is performed in thesame way and corrections are made.
0.1 mol/L ceric ammonium sulfate solution 1 mL = 9.012 mg C11 H16 O2(b) Standard of storage method

It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of butylatedhydroxyanisole are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of butylatedhydroxyanisole is applied mutatis mutandis.

(11) Propionic acid
A. Raw material for manufacturing(a) Compositional standards
Contents: When this product is determined, it contains 99.0 % or more of propinic acid
(C3H6O2).
Physical and chemical properties:
i. This product is a clear liquid with specific odor.
ii. This product is easy to extremely dissolve in water and easy to dissolve in alcohol,
ether, chloroform and acid.
iii. The pH of solution (1 → 100) of this product is 2.0~2.5.
Confirmation test:
i. The solution (1 → 10) of this product is acidic.
ii. When 1 mL of this product is added with 1 mL of ethanol and 3 drops of sulfuric acidand heated in a water bath, it emits the odor of ethyl propionate.
Purity test:
i. Residue on evaporation: When 50 mL of this product is evaporated on a water bath,and dried to a constant weight at 105~110 °C, the residue shall be 5 mg or less (0.01 %or less).
ii. Arsenic: 1 mL of this product is weighed and a sample solution is prepared by MethodNo.2 of the arsenic test method. The arsenic test is performed by the method using
device A. The color of absorbing solution shall not be darker than the standard color (2µg/mL or less).

248

Provisional Translation from Japanese Originaliii. Heavy metal: 2 mL of this product is weighed and a sample solution is prepared by
Method No.1 of the heavy metal test method. A control solution is prepared using 2
mL of lead standard solution. When the sample and control solution are tested forheavy metal, the color of the sample solution shall not be darker than the standard
color (10 µg/mL or less).
Assay: Approximately 3 g of this product is weighed to the digits of 0.01 g and the value isrecorded. It is dissolved with 100 mL of newly boiled and cooled water and titrated with
1 mol/L sodium hydroxide solution (indicator: 3 drops of phenolphthalein test solution).
A blank test is performed in the same way and corrections are made.
1 mol/L sodium hydroxide solution 1 mL = 74.08 mg C3H6O2(b) Standard of storage method
It shall be stored in a airtight container.
B. Preparation (Part1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of propionic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of propionic acid isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is piece or powder of the mixture of the raw material for manufacturing of
propionic acid and fillers.
Contents: When this product is determined, it conteins propinic acid equivalent to95~105 % of declared content.
Confirmation test: According to the amount of this product on the label, the amountcontaining approximately 2 g of the raw material for manufacturing of propionic acid isweighed, added with 20 mL of ethanol, boiled, and filtered. When 1 mL of the filtrate ismeasured, added with 3 drops of sulfuric acid and heated in a water bath, it emits the odorof ethyl propionate.
Assay: The amount of this product containing approximately 2.0 g of propionic acid
(C3H6O2) is weighed to three significant digits and the value is recorded. It is placed in a
1 L distillation flask and added with 200 mL of water, 80 g (79.5~80.4 g) of sodium
chloride, 10 mL of 10% phosphoric acid solution, and a drop of silicone emulsion. The 1
L distillation flask is connected to a distillation device. A 500 mL volumetric flask is used
as a receiver, added with 20 mL of water, and the bottom of the condenser is immersed in249

Provisional Translation from Japanese Original

this solution. When the distillate from water vapor approaches 500 mL, the distillation is
stopped and water is added to the graduation line to make 500 mL. Ater shakeing it well,
50 mL of it is measured using a volumetric pipette and titrated with 0.1 mol/L sodiumhydroxide solution (indicator: 3 drops of phenolphthalein test solution). A blank test is
performed in the same way and corrections are made.
0.1 mol/L sodium hydroxide solution 1 mL = 7.408 mg C3H6O2(b) Standard of storage method
It shall be stored in a capped container.

(12) Calcium propionate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried at 120 °C for 2 hours and then determined, it contains
98.0 % or more of calcium propionate (C6H10CaO4).
Physical and chemical properties:
i. This product is white crystals, granules or powder, with no odor, or a slight specificodor.
ii. This product is easy to dissolve in water.
Confirmation test:
i. When 0.5 g (0.45~0.54 g) of this product is dissolved with 5 mL of water, and addedwith 5 mL of dilute sulfuric acid and heated, it emits a specific odor.
ii. A solution of this product in water (1 → 10) gives the qualitative reaction of calcium
salt i., ii., and iii..
Purity test:
i. Water-insoluble matter: 10.0 g (9.95~10.04 g) of this product is weighed, added with
100 mL of water, allowed to stand for an hour with occasional shaking, and filteredwith a glass filter (G4). When the residue is washed with 30 mL of water and dried at
180 °C for 4 hours, its amount shall be 0.015 g or less.
ii. Acid and alkali: 2.0 g (1.95~2.04 g) of this product is weighed, dissolved with 20 mLof newly boiled and cooled water, added with 2 drops of phenolphthalein test solution
to prepare a sample solution.When a sample solution is colorless, added with 0.3 mL
of 0.1 mol/L sodium hydroxide solution, it changes red. When a sample solution is redcolor, added with 0.3 mL of 0.1 mol/L hydrochloric acid, the color disappears.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No.1 of the heavy metal test method. A control solution is250

Provisional Translation from Japanese Original

prepared using 2 mL of lead standard solution. When the sample and control solution
are tested for heavy metal, the color of the sample solution shall not be darker than thestandard color (20 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No.1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 9.5 % or less (1 g, 120 °C, 2 hours)
Assay: This product is dried at 120 °C for 2 hours, approximately 1 g of it is weighed to thedigits of 0.01 g, and the value is recorded. It is dissolved with water, transferred to a 100mL volumetric flask, and added with water to graduation line to make 100 mL. 25 mL ofthis solution is measured using a volumetric pipette, added with 75 mL of water and 15
mL of potassium hydroxide solution (1 → 10), allowed to stand for approximately 1minute, added with 0.1 g (0.05~0.14 g) of NN indicator, and titrated with 0.05 mol/L
ethylenediaminetetraacetic acid disodium solution within 30 seconds. In this case, the end
point of the titration is the point at which the color of the solution changes from reddish
violet to blue. A blank test is performed in the same way and corrections are made.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 9.311 mgC6H10CaO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is water-soluble liquid, a mixture of the raw material for manufacturing of
calcium propionate and water.
Contents: When this product is determined, it contains calcium propionate (C6H10CaO4)
corresponding to 95~105 % of the amount of the label.
Confirmation test:
i. When, according to the amount of this product on the label, the amount containing
approximately 0.5 g of the raw material for manufacturing of calcium propionate isweighed and heated with 5 mL of dilute sulfuric acid, it emits a specific odor.
ii. This product gives the qualitative reaction of calcium salt i., ii., and iii..
Assay: According to the amount of this product on the label, the amount containing
approximately 1 g of the raw material for manufacturing of calcium propionate isweighed to three significant digits and the value is recorded. It is placed in a 100 mL
volumetric flask and added with water to the graduation line to make 100 mL. 25 mL ofthis solution is measured using a volumetric pipette, added with 75 mL of water and 15

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Provisional Translation from Japanese Original

mL of potassium hydroxide solution (1 → 10), allowed to stand for approximately 1minute, added with 0.1 g (0.05~0.14 g) of NN indicator, and titrated with 0.05 mol/L
ethylenediaminetetraacetic acid disodium solution within 30 seconds. In this case, the end
point of the titration is the point at which the color of the solution changes from reddish
violet to blue. A blank test is performed in the same way and corrections are made.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 9.311 mg C6H10CaO4(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium
propionate is applied mutatis mutandis.
C. Preparation (Part 2 powder)(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium propionate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium
propionate is applied mutatis mutandis.

(13) Sodium propionate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried at 105 °C for 1 hours and then determined, it contains
99.0 % or more of sodium propionate (C3H5NaO2).
Physical and chemical properties:
i. This product is white crystals, granules or crystalline powder, with no odor, or a slightspecific odor.
ii. This product is easy to dissolve in water and easy to slightly dissolve in ethanol.
iii. This profuct is hygroscopic.
Confirmation test:
i. The confirmation test i. of calcium propionate is applied mutatis mutndis.
ii. The solution (1 → 10) of this product gives the qualitative reaction of sodium salt i.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is weighed,dissolved with 20 mL of water, the resulting solution shall be colorless and theturbidity shall be delicately slight cloudiness or clearer.

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Provisional Translation from Japanese Original

ii. Acid and alkali: 2.0 g (1.95~2.04 g) of this product is weighed, dissolved with 20 mLof newly boiled and cooled water, added with 2 drops of phenolphthalein test solution
to prepare a sample solution. When a sample solution is colorless, added with 0.3 mL
of 0.1 mol/L sodium hydroxide solution, it changes red. When a sample solution is redcolor, added with 0.3 mL of 0.1 mol/L hydrochloric acid, the color disappears.
iii. Heavy metal: 1.0 g (0.95~1.04) of this product is weighed and a sample solution isprepared by Method No.1 of the heavy metal test method. A control solution isprepared using 2.0 mL of lead standard solution. When the sample and control solution
are tested for heavy metal, the color of the sample solution shall not be darker than thestandard color (20 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04) of this product is weighed and a sample solution isprepared by Method No.1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 5.0 % or less (1 g, 105 °C, 1 hours)
Assay: This product is dried at 105 °C for 1 hours, approximately 0.2 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is added with 40 mL of glacial acetic
acid for nonaqueous titration, warmed to be dissolved, allowed to cool, and titrated with
0.1 mol/L perchloric acid (indicator: 3 drops of methylrosanilinium chloride test
solution). A blank test is performed in the same way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 9.606 mg C3H5NaO2(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium propionate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
propionate is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
The product is powder of the mixture of the raw material for manufacturing of sodium
propionate and fillers.
Contents: When this product is determined, it contains sodium propionate (C3H5NaO2)
corresponding to 90~110 % of of the amount on the label.

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Provisional Translation from Japanese OriginalConfirmation test:
i. 0.5 g (0.45~0.54 g) of this product is added with 5mL of water, stirred well andfiltered.When the filtrate is added with 5 mL of dilute sulfuric acid and heated, it emitsa specific odor.
ii. When 1 g (0.5~1.4 g) of this product is added with 10 mL of water, stirres, andfiltered, the filtrate gives the qualitative reaction of sodium salt i..
Assay: This product is dried at 105 °C for an hour, approximately 5 g of it is weighed to thedigits of 0.01 g, and the value is recorded. It is added with approximately 60 mL of water,
stirred well, and filtered. The filtrate is collected directly in a 100 mL volumetric flask.
The beaker used to dissolve the sample is washed with approximately 30 mL of water andthe solids on the filter are washed with the washings. The filtrate and the washings are
mixed and added with water to the 100 mL volumetric flask to the graduation line. This isused as a sample solution.
20 mL of the sample solution is measured using a volumetric pipette, and placed in aporcelain or platinum pot with a diameter of 50~55 mm, gently heated to evaporate
water, and then gradually increasing temperature, heated for approximately 2 hours to becompletely carbonized. The heating temperature shall be the temperature at which theheated pot is dark red (300~400 °C). In this case, the flame of the burner shall not contact
the carbide. After cooling, the carbide in the pot is crushed well with a glass bar and put
in a beaker along with the pot. It is added with approximately 50 mL of water, addedwith 50 mL of 0.25 mol/L sulfuric acid, covered with a watch glass, heated on a water
bath for an hour, and filtered. If the filtrate is colored, the sample is newly weighed andsufficiently carbonized. The residues in the beaker and the pot and on the filter are
washed well with warm water. When the washings do not change blue litmus paper tored, the washings are mixed with the filtrate and excessive acid is titrated with 0.5 mol/L
sodium hydroxide solution (indicator: 3 drops of methyl red test solution). A blank test is
performed in the same way.
0.25 mol/L sulfuric acid 1 mL = 48.03 mg C3H5NaO2(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
propionate is applied mutatis mutandis.

(14) Propylene glycol
A. Raw material for manufacturing(a) Compositional standards

254

Provisional Translation from Japanese OriginalContents: When this product is determined, it contains 97.5 % or more of propinic acid
(C3H8O2).
Physical and chemical properties:
i. This product is colorless, transparent, syrupy liquid, with no odor or a slight, specificodor, and with a slight bitter taste.
ii. This product mix with water, aceton, ethanol or chloroform.
iii. This product is easy to dissolve in ether.
iv. This product is hygroscopic.
Confirmation test:
i. 0.5 g (0.45~0.54 g) of this product is added with 5 mL of pyridine and 3.6 g (3.55~3.64g) of triphenyl chloromethane, heated on a water bath for an hour with a reflux
condenser, allowed to cool, dissolved with 100 mL of warm acetone, added with 0.1 g(0.05~0.14 g) of activated charcoal, shaken up well and filtered. The filtrate isevaporated to dryness on a water bath to 50 mL and allowed to stand in a refrigerator
overnight. The produced crystals are collected by filtration and recrystallized threetimes with very little acetone until the odor of pyridine disappears. When these aredried at 105 °C for 30 minutes, the melting point is 173~179 °C.
ii. When 1 mL of this product is added with 0.5 g (0.45~0.54 g) of potassium hydrogensulfate and gently heated, it emits a fruit-like odor.
Purity test:i. Specific gravity: The specific gravity of this product d20
20 shall be 1.036~1.040.
ii. Acid: When 10 mL of this product is added with 50 mL of newly boiled and cooled
water to mix well, added with 5 drops of phenolphthalein test solution and 0.30 mL of
0.1 mol/L sodium hydroxide solution, the resulting solution shall be red.
iii. Chloride: 2.0 g (1.95~2.04 g) of this product is weighed and a sample solution isprepared by chloride test method. A control solution is used 0.4mL of 0.01 mol/Lhydrochloric acid. When the sample and control solution are tested for chloride, theturbidity of the sample solution shall not be higher than that of control solution(0.007 % or less).
iv. Heavy metal: 5.0 g (4.95~5.04 g) of this product is weighed and a sample solution isprepared by Method No.1 of the heavy metal test method. A control solution isprepared using 2.5 mL of lead standard solution. When the sample and control solution
are tested for heavy metal, the color of the sample solution shall not be darker than thestandard color (5 mg/kg or less).
v. Arsenic: 0.7 g (0.65~0.74 g) of this product is weighed and a sample solution isprepared by Method No.1 of the arsenic test method. The arsenic test is performed by255

Provisional Translation from Japanese Original

the method using device A. The color of absorbing solution shall not be darker than
the standard color (3 mg/kg or less).
vi. Glycerin and ethylene glycol: Approximately 1 g of this product is weighed to thedigits of 0.01 g and the value is recorded. It is added with water to make 1,000 mL, ofwhich 13 mL is measured, added with 0.2 g (0.15~0.24 g) of potassium periodate and
1 mL of sulfuric acid, and further added with 50 mL of water and distilled at a rate of
3-5 mL/min. It is continued distillation until the residual solution is about 1 mL.The
distillate is collected in a receiver cooled in ice water. The distillate is added withwater to make 500 mL to prepare a sample solution. When 1 mL of the sample
solution is measured, added with 0.1 g (0.05~0.14 g) of chromotropic acid and 5 mL of
sulfuric acid, heated in a water bath for 30 minutes, cooled, and added with water tomake 250 mL, the color of the solution shall not be darker than a solution obtained by
the same procedure for preparation of a sample solution using 1 mL of formaldehyde
standard solution.
Water content: 0.20 % or less (direct titration)
Ignition residue: Approximately 20 g of this product is weighed to the digits of 0.1 g and thevalue is recorded. It is gently heated to boil and then the heating is stopped. It is ignited
within 30 seconds to burn and allowed to cool. When the residue is moisturized with 0.2
mL of sulfuric acid and tested by the ignition residue test method, the amount shall be
0.05 % or less.
Distillation test: 184~189 °C, 95 v/v% or more
Assay: Approximately 1 g of this product is weighed to the digits of 0.01 g and the value isrecorded. It is dissolved with water, transferred to a 250 mL volumetric flask, and addedwith water to the graduation line to make 250 mL. 10 mL of this solution is measured
using a volumetric pipette, transferred to a stoppered flask, added with 10 mL of sodium
periodate test solution using a volumetric pipette, further added with 4 mL of sulfuric
acid (1 → 2), shaken up, and allowed to stand for 40 minutes. It is added with 5 g (4.5～
5.4 g) of potassium iodide, sealed with a stopper within 30 seconds, shaken up well,allowed to stand in a dark place for 5 minutes, added with 50 mL of 0.1 mol/L sodium
thiosulfate solution using a volumetric pipette, and titrated with 0.1 mol/L sodium
thiosulfate solution (indicator: 1 mL of starch test solution). Separately, a blank test is
performed in the same way.
0.1 mol/L sodium thiosulfate solution 1 mL = 3.805 mg C3H8O2(b) Standard of storage method
It shall be stored in a capped container.

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Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of proplene glycol areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of proplene glycol
is applied mutatis mutandis.

(15) Sodium polyacrylate
A. Raw material for manufacturing(a) Compositional standards
Physical and chemical properties: This product is white powder with no odor.
Confirmation test:
i. 0.2 g (0.15~0.24 g) of this product is added with 100 mL of water, then stirred well todissolve. This is used as a sample solution. When 10 mL of this sample solution isadded with 1 mL of calcium chloride test solution and shaken up, white precipitation is
generated within 30 seconds.
ii. The residue obtained by ashing of this product gives the qualitative reaction of sodium
salt.
Purity test:
i. Free alkali: 0.2 g (0.15~0.24 g) of this product is added with 60 mL of water, stirred
well to dissolve, added with 3 mL of calcium chloride test solution, heated on a water
bath for approximately 20 minutes, is allowed to cool and is filtered. The residue on
the filter is washed with water, and the washings are mixed with the filtrate and addedwith water to make 100 mL to prepare a sample solution. When 50 mL of the sample
solution is measured and added with 2 drops of phenolphthalein test solution, it shallnot be red.
ii. Sulfate: 20 mL of the sample solution i. is measured, added with 1 mL of dilutehydrochloric acid and water to make 50 mL. This is used as a sample solution. Whensulfate is tested using a control solution prepared with 0.4 mL of 0.005 mol/L sulfuric
acid using the sulfate test method, the turbidity of the sample solution shall not behigher than that of the control solution (0.49 % or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No.2 of the heavy metal test method. A control solution isprepared using 2.0 mL of lead standard solution. When the sample and control solution

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Provisional Translation from Japanese Original

are tested for heavy metal, the color of the sample solution shall not be darker than thestandard color (20 mg/kg or less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No.3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
v. Residual monomer: Approximately 1 g of this product is weighed to the digits of 0.01
g and the value is recorded. It is placed in a 300 mL iodine bottle, added with 100 mLof water, shaken up occasionally, and allowed to stand for 24 hours to dissolve. 10 mLof potassium bromate-potassium bromide test solution using a volumetric pipette,
shaken up well, quickly added with approximately 10 mL of hydrochloric acid, and astopper is closed within 30 seconds to be shaken up well. Approximately 20 mL ofpotassium iodide test solution is put in the upper part of the iodine bottle and allowed
to stand in a dark place for 20 minutes. Then, the stopper is loosened, the potassium
iodide test solution is poured, and the stopper is closed within 30 seconds to be shaken
up well. Then, it is titrated with 0.1 mol/L sodium thiosulfate solution (indicator: 2 mLof starch test solution). Separately, a blank test is performed in the same way. The
amount of the residual monomer calculated using the following equation shall be 1 %or less.
Amount of residual monomer=

0.0047 x (a- b)
x 100 (%)
Amount of the sample weighed (g)a: Consumed volume of 0.1 mol/L sodium thiosulfate solution in the blank test(mL)
b: Consumed volume of 0.1 mol/L sodium thiosulfate solution in the main test (mL)vi. Low polymer: 2 g (1.95~2.04 g) of this product is added with 200 mL of water,
shaken up occasionally, allowed to stand for 24 hours to dissolve. In this solution 50
mL of 10 mol/L hydrochloric acid is added dropwise with stirring, warmed in a water
bath at 40 °C for 30 minutes with stirring and then is allowed to stand for 24 hours.
The generated precipitation is filtered and the filtrate is added with a drop ofphenolphthalein test solution, added with sodium hydroxide solution (2 → 5) until thefiltrate is slightly red, and then added with hydrochloric acid (1 → 30) by dropping
until the red color disappears. 200 mL of water is added, and 25 mL of calcium
chloride test solution is dropped with stirring and heated in a water bath atapproximately 40 °C for 30 minutes with stirring. The generated precipitation isfiltered by suction using a glass filter (1G4) whose mass is known. The residue iswashed three times with approximately 10 mL each of water, dried at 105 °C for 4258

Provisional Translation from Japanese Original

hours. The amount of the lower polymer calculated using the following equation shallbe 5 % or less.
Low polymer=

Residue (g) x 1.0324
x 100 (%)
Amount of the sample weighed (g)Loss on dring: 10 % or less (1.5g, 105°C, 4 hours)
Ignition residue: After drying 76% or less (1 g)(b) Standard of storage method
It shall be stored in a capped container avoiding a moisture.
B. Preparation (Part1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium
polyacrylate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
polyacrylate is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
The product is powder of the mixture of the raw material for manufacturing of sodium
polyacrylate and fillers.
Confirmation test: According to the amount of this product on the label, the amountcontaining approximately 0.2 g of the raw material for manufacturing of sodium
polyacrylate is weighed and dried at 140 °C for an hour. This preparation is placed in a
separatory funnel, added with 100 mL of carbon tetrachloride, vigorously shaken up, andallowed to stand for 5 minutes to separate the bottom precipitation layer (layer containing
sodium polyacrylate). The separated layer is added with 50 mL of a mixture of ethanol
and water (1:1), stirred well, and allowed to stand for a few minutes, and the upper layer
is removed by suction. At this time, sodium polyacrylate is separated to the middle zonal
layer. Then the middle layer is filtered by suction using a glass filter (G3). The
precipitation in the bottom layer is discarded. Then the residue on the funnel istransferred to a 200 mL erlenmeyer flask, dried, added with 100 mL of water, and stirred
well to dissolve. The confirmation test i. and ii. of the raw material for manufacturing of
sodium polyacrylate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
polyacrylate is applied mutatis mutandis.

259

Provisional Translation from Japanese Original(16) Polyoxyethylene Glycerol Fatty Acid Ester
A. Raw material for manufacturing(a) Compositional standards
This product is a polyoxyethylene adduct of an ester of fatter acids derived from animal
fat and plant oil and glycerin.
Physical and chemical properties: This product is a white to yellow-brown liquid,semiliquid, or waxy blocks, with no odor or a slight, specific odor.
Confirmation test:
i. When 0.5 g (0.45~0.54 g) of this product is added with 5 mL of absolute ethanol,
heated to dissolve, added with 5 mL of dilute sulfuric acid, heated in a water bath for
30 minutes, and then cooled, oil drops or a white to yellow-white solid is deposited.
When the oil drops or solid is separated, added with 5 mL of ether and shaken up, itdissolves.
ii. When 1 g (0.5~1.4 g) of this product is added with 20 mL of water, warmed, shaken
up well, added with 10 mL of ammonium thiocyanate-cobalt nitrate test solution,
shaken up well, added with 10 mL of chloroform, shaken up, and allowed to stand, thechloroform layer is blue.
Purity test:i. Acid value: Approximately 5 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is added with 100 mL of a solution of ethanol and ether (1:1),
warmed to dissolve, allowed to cool, added with 1 mL of phenolphthalein test solution,and titrated with 0.1 mol/L potassium hydroxide-ethanol solution. Separately, a blanktest is performed in the same way. The acid value calculated by the following equation
shall be 6 or less.

Acid value

Consumed volume of 0.1 mol/L
potassium hydroxide-ethanol solution (mL) × 5.611=
Amount of the sample weighed (g)ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).

260

Provisional Translation from Japanese Originaliv. 1,4-Dioxane: The peak height of this product obtained from the test by the 1,4Dioxane test method shall not be higher than that of 0.2 mL of the measurement
standard solution of 1,4-Dioxane (10 mg/kg or less).
Ignition residue: 1.5 % or less (1 g)(b) Standard of storage method
It shall be stored in a lightproof sealed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of polyoxyethylene
glycerolin fatty acid ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of polyoxyethylene
glycerolin fatty acid ester is applied mutatis mutandis.

(17) Polyoxyethylene Sorbitan Fatty Acid Ester
A. Raw material for manufacturing(a) Compositional standards
This is a polyoxyethylene adduct of an ester of fatter acids derived from animal fat andplant oil and sorbitan.
Physical and chemical properties: This product is a yellow to brown liquid, semiliquid or
waxy blocks.
Confirmation test:
i. When 0.5 g (0.45~0.54 g) of this product is added with 5 mL of absolute ethanol,
heated to dissolve, added with 5 mL of dilute sulfuric acid, heated in a water bath for
30 minutes, and then cooled, oil drops or a white to yellow-white solid is deposited.
When the oil drops or solid is separated, added with 5 mL of ether and shaken up, itdissolves.
ii. When 1 g (0.95~1.04 g) of this product is added with 20 mL of water, warmed, shaken
up well, allowed to cool, added with 10 mL of ammonium thiocyanate-cobalt nitrate
test solution, shaken up well, added with 10 mL of chloroform, shaken up, and allowed
to stand, the chloroform layer is blue.
Purity test:i. Acid value: Approximately 5 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is added with 100 mL of a solution of ethanol and ether (1:1),
warmed to dissolve, allowed to cool, added with 1 mL of phenolphthalein test solution,

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Provisional Translation from Japanese Original

and titrated with 0.1 mol/L potassium hydroxide-ethanol solution. Separately, a blanktest is performed in the same way. The acid value calculated by the following equation
shall be 6 or less.

Acid value =Consumed volume of 0.1 mol/L
potassium hydroxide-ethanol solution (mL) × 5.611
Amount of the sample weighed (g)ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
iv. 1,4-Dioxane: Approximately 5 g of this product is weighed and the peak height of thisproduct obtained from the test by the 1,4-Dioxane test method shall not be higher thanthat of 0.2 mL of the measurement standard solution of 1,4-Dioxane (10 mg/kg or
less).
Ignition residue: 1.5 % or less (1 g)(b) Standard of storage method
It shall be stored in a lightproof sealed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of polyoxyethylene
sorbitan fatty acid ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of polyoxyethylene
sorbitan fatty acid ester is applied mutatis mutandis.

(18) L-ascorbic Acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (silica gel) for 24 hours and determined,
it contains 99.9 % or more of L-ascorbic acid (C6H8O6).

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Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor and with a sour taste.
ii. This product is easy to dissolve in water, hard to slightly dissolve in ethanol, andhardly dissolves in ether, chloroform, benzene, or petroleum ether.
iii. The pH of the solution of this product in water (1 → 50) is 2.4~2.8.
iv. Melting point: About 190 °C (decomposition)
Confirmation test:
i. 5 mL each of a solution of this product in water (1 → 50) is measured. When 1 drop ofpotassium permanganate test solution is added to the solution and when 1~2 drops of
2,6-dichlorophenolindophenol sodium test solution is added to it, the color of theboth test solutions disappears within 30 seconds.
ii. 0.1 g (0.05~0.14 g) of this product is dissolved in 100 mL of metaphosphoric acidsolution (1 → 50) and 5 mL of this solution is measured. Iodine test solution is
dropped in the solution until it is slightly yellow and then a drop of copper sulfate
solution (1 → 1,000) and a drop of pyrrole are added to it. When the resulting solution
is warmed in a water bath at 50 °C for 5 minutes, it is blue.
Purity test:i. Specific rotation: Approximately 2.5 g of this product is weighed to the digits of 0.01 g
and the value is recorded. It is dissolved in water to make 25 mL. The optical rotationof this solution shall be [α]20
D = +20.5° ~ +21.5°.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 0.20 % or less (1 g, silica gel, 24 hours)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried in a desiccator (silica gel) for 24 hours, approximately 0.2 g ofit weighed to the digits of 0.001 g, and the value is recorded. It is dissolved with 50 mL of
metaphosphoric acid solution (1 → 50) and titrated with 0.1 mol/L iodine solution(indicator: 1 mL of starch test solution).
0.05 mol/L iodine solution 1 mL = 8.806 mg C6H8O6(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation (Part 1)(a) Compositional standards

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The compositional standards of the raw material for manufacturing of L-ascorbic acidare applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-ascorbic acidis applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof L-ascorbic acid and carrier.
Content: When this product is determined, it contains L-ascorbic acid (C6H8O 6)
corresponding to 90~120 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 g of the
raw material for manufacturing of L-ascorbic acid is weighed, added with 50 mL ofwater and for a preparation containing hydrogenated oils, higher saturated fatty acids,
fatty acids, vegetable oil or animal fats added with 15 mL of chloroform, shaken up,
and allowed to stand. The aqueous layer is filtered to prepare a sample solution and 5
mL of this solution is measured. The confirmation test i. of the raw material for
manufacturing of L-ascorbic acid is hereinafter applied mutatis mutandis.
ii. 1 mL of the test solution i. is added with 19 mL of metaphosphoric acid solution (1 →50) to mix them. 5 mL of this solution is measured and the confirmation test ii. of the
raw material for manufacturing of L-ascorbic acid is hereinafter applied mutatis
mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forothers.
Method No. 1: The amount of this product containing approximately 0.1 g of L-ascorbic
acid (C6H8O6) is weighed to three significant digits and the value is recorded. It is
added with 50 mL of chloroform, shaken up, and filtered using a glass filter (G4). The
residue on the filter is washed three times with 20 mL each of chloroform. When thechloroform odor on the residue disappears, the residue is filtered by extracting with
190 mL of metaphosphoric acid-acetic acid test solution. The filtrate is placed in a 200mL volumetric flask, added with metaphosphoric acid-acetic acid test solution to thegraduation line to make 200 mL. This is used a sample solution. 2 mL of the sample
solution is measured using a volumetric pipette, added with 8 mL of metaphosphoric
acid-acetic acid test solution and 2 mL of hydrogen peroxide test solution, shaken up,

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and titrated with 2,6-dichlorophenolindophenol sodium solution for titration until pinkcolor of the solution persists for 5 seconds. A blank test is performed in the same way
and corrections are made.
2,6-dichlorophenolindophenol sodium test solution for titration 1 mL = A mg C6H8O6
A: Amount of L-ascorbic acid (C6H8O6) corresponding to 1 mL of 2,6dichlorophenolindophenol sodium test solution for titration
The reference standard of L-ascorbic acid is dried in a desiccator (silica gel) for 24 hours.
Approximately 0.05 g of it is weighed to the digits of 0.1 mg and the value is recorded.
It is dissolved with metaphosphoric acid-acetic acid test solution and transferred to a
100 mL volumetric flask, and further added with metaphosphoric acid-acetic acid test
solution to the graduation line to make 100 mL, and hereinafter subjected to the sameprocedure for the sample solution to obtain A.
Method No. 2: The amount of this product containing approximately 0.1 g of L-ascorbic
acid (C6H8O6) is weighed to three significant digits and the value is recorded. It is
added with 150 mL of metaphosphoric acid-acetic acid test solution, vigorously
shaken up and filtered. The residue on the filter is washed twice with 15 mL each of
metaphosphoric acid-acetic acid test solution. The filtrate and the washings are mixed,
the mixture is placed in a 200 mL volumetric flask and added with metaphosphoric
acid-acetic acid test solution to the graduation line to make 200 mL, and hereinafter
the method No. 1 is applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof sealed container.

(19) Calcium L-Ascorbate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.0 % or more of Calcium Lascorbate (C12H14CaO12·2H2O).
Physical and chemical properties:
i. This product is white to yellowish-white crystal powder with no odor.
ii. This product is easy to dissolve in water, and hardly dissolves in acetone, ether ormethanol.
Confirmation test:
i. When 2 ml of the solution of this product in water (1 → 100) is measured and addedwith 4 mL of 0.1 mol/L hydrochloric acid, 5~6 drops of sodium nitroprusside test265

Provisional Translation from Japanese Original

solution and 2 drops of 1 mol/L sodium hydroxide test solution, the resulting solution
is blue within 30 seconds.
ii. 0.1 g (0.05~0.14 g) of this product is dissolved with 100 mL of metaphosphoric acidsolution (1 → 50). When iodine test solution is dropped in 5 mL of this solution until
the solution is slightly yellow, added with a drop of copper sulfate solution (1 →1,000) and a drop of pyrrole, heated at 50 °C for 5 minutes, it is blue to blue-green.
iii. The solution of this product in water (1 → 10) gives the qualitative reaction of
calcium salt.
Purity test:i. Specific rotation: Approximately 2.5 g of this product is weighed to the digits of 0.01 g
and the value is recorded. It is dissolved in water to make 100 mL. The rotation of thissolution shall be [α]20
D = +95° ~ +97°.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Ignition residue: 30.0~33.0 % (1 g)Assay: Approximately 0.2 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 50 mL of metaphosphoric acid solution (1 → 50) andtitrated with 0.05 mol/L iodine solution (indicator: 1 mL of starch test solution).
0.05 mol/L iodine solution 1 mL = 10.66 mg C12H14CaO12·2H2O(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium Lascorbate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium Lascorbate is applied mutatis mutandis.

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C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof calcium L-ascorbate and carrier.
Content: When this product is determined, it contains calcium L-ascorbate(C12H14CaO12·2H2O) corresponding to 90~120 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.15 g of
the raw material for manufacturing of calcium L-ascorbate is weighed and added with
15 mL of water, and for a preparation containing hydrogenated oils, higher saturated
fatty acids, fatty acids, vegetable oil or animal fats added with 5 mL of chloroform, isshaken up and allowed to stand. The aqueous layer is filtered to prepare a sample
solution. 2 mL of this solution is measured and the confirmation test i. of the raw
material for manufacturing of calcium L-ascorbate is hereinafter applied mutatis
mutandis.
ii. 1 mL of the sample solution i. is added with 9 mL of metaphosphoric acid solution (1→ 50) to mix them. 5 mL of the solution is measured and the confirmation test ii. forthe raw material for manufacturing of calcium L-ascorbate is hereinafter appliedmutatis mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forothers.
Method No. 1: The amount of this product containing approximately 0.1 g of calcium Lascorbate (C12H14CaO12·2H2O) is weighed to three significant digits and the value isrecorded. It is added with 50 mL of chloroform, shaken up, and filtered using a glass
filter (G4). The residue on the filter is washed three times with 20 mL each ofchloroform. When the chloroform odor on the residue disappears, the residue isfiltered by extracting with approximately 190 mL of metaphosphoric acid-acetic acidtest solution. The filtrate is placed in a 200 mL volumetric flask, added withmetaphosphoric acid-acetic acid test solution to the graduation line to make 200 mL.
This is used as a sample solution. 2 mL of the sample solution is measured using a
volumetric pipette, added with 8 mL of metaphosphoric acid-acetic acid test solution
and 2 mL of hydrogen peroxide test solution, shaken up, and titrated with 2,6dichlorophenolindophenol sodium solution for titration until a pink color of thesolution persists for 5 seconds. A blank test is performed in the same way andcorrections are made.

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2,6-dichlorophenolindophenol sodium test solution for titration 1 mL = (A × 1.210)
mg C12H14CaO12·2H2O
A: Amount (mg) of L-ascorbic acid (C6H8O6) corresponding to 1 mL of 2,6dichlorophenolindophenol sodium test solution for titration
The reference standard of L-ascorbic acid is dried in a desiccator (silica gel) for 24hours. Approximately 0.05 g of it is weighed to the digits of 0.1 mg and the value isrecorded. It is dissolved with metaphosphoric acid-acetic acid test solution andtransferred to a 100 mL volumetric flask, and added with metaphosphoric acid-acetic
acid test solution to the graduation line to make 100 mL, and hereinafter subjected tothe same procedure for the sample solution to obtain A
Method No. 2: The amount of this product containing approximately 0.1 g of calcium Lascorbate (C12H14CaO12·2H2O) is weighed to three significant digits and the value isrecorded. It is added with 150 mL of metaphosphoric acid-acetic acid test solution,
vigorously shaken up and filtered. The residue on the filter is washed twice with 15
mL each of metaphosphoric acid-acetic acid test solution. The filtrate and the washings
are mixed, the mixture is placed in a 200 mL volumetric flask and added withmetaphosphoric acid-acetic acid test solution to the graduation line to make 200 mL,
and hereinafter the method No. 1 is applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof sealed container.

(20) Sodium L-Ascorbate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a vacuum desiccator (silica gel) for 24 hours anddetermined, it contains 99.0 % or more of sodium L-ascorbate (C6H7NaO6).
Physical and chemical properties:
i. This product is white to yellowish-white crystal powder with no odor.
ii. This product is easy to dissolve in water, and hardly dissolves in ethanol.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 50) is added with 1-2 drops of
2,6-dichlorophenolindophenol sodium test solution, the color of the test solution
disappears within 30 seconds.
ii. 0.1 g (0.05~0.14 g) of this product is dissolved in 100 mL of metaphosphoric acidsolution (1 → 50). When 5 mL of this solution is dropped with iodine test solution

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until the solution is slightly yellow, added with a drop of copper sulfate solution (1 →1,000) and a drop of pyrrole and warmed at 50 °C for 5 minutes, it is blue to bluegreen.
iii. The solution of this product in water (1 → 10) gives the qualitative reaction of sodium
salt.
Purity test:i. Specific rotation: Approximately 2.5 g of this product is weighed to the digits of 0.01 g
and the value is recorded. It is dissolved in water to make 25 mL. The rotation of thissolution shall be [α]20
D = +103.0° ~ +108.0°.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Loss on drying: 0.50 % or less (1 g, reduced pressure, silica gel, 24 hours)
Assay: This product is dried in a vacuum desiccator (silica gel) for 24 hours, andapproximately 0.2 g of it is weighed to the digits of 0.001 g, and the value is recorded.
When it is dissolved with 50 mL of metaphosphoric acid solution (1 → 50) and titrated
with 0.05 mol/L iodine solution (indicator: 1 mL starch test solution).
0.05 mol/L iodine solution 1 mL = 9.906 mg C6H7NaO6(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium Lascorbiate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium Lascorbiate is applied mutatis mutandis.

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Provisional Translation from Japanese Original(21) Sodium-calcium-L-Ascorbic acid-2-Phosphate ester
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 60.0 % or more of sodium calcium Lascorbic acid-2-phosphate ester ((C6H6O9P) 2 Na4 Ca).
Physical and chemical properties: This product is white to grayish brown powder.
Confirmation test:i. 2 mg (1.5~2.4 mg) of this product is added with 0.1 mol/L hydrochloric acid solution
to make 100 mL. In the measurement of the absorption spectrum of this solution, theabsorption maximum is at the wavelength of 235~239 nm.
ii. 90 mg (89.5~90.4 mg) of this product is weighed, stirred with 20 mL of water, andcentrifuged for 5 minutes at 3,000 rpm. The supernatant is used as a sample solution.
Separately, 5 mg (4.5~5.4 mg) of the reference standard of L-ascorbic acid-2phosphate ester tris cyclohexylammonium is weighed and dissolved with 1 mL ofwater to prepare a standard solution. 5 µL each of the sample and standard solution isspotted on a thin layer plate prepared using cellulose powder thin-layer
chromatography (with a fluorescent agent). Then, they are developed to 10 cm with thedeveloping solvent, a mixture of absolute ethanol, water and ammonia solution(20:10:3), and the thin layer plate is air dried. When it is irradiated with ultraviolet
light (dominant wavelength: 254 nm), the spots obtained from the sample and standard
solutions are dark blue and their Rf values are equal.
Purity test:
pH: The pH of the solution of this product in water (1 → 50) is 7.5~9.5.
Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution is preparedby Method No. 3 of the arsenic test method. The arsenic test is performed by themethod using device A. The color of absorbing solution shall not be darker than thestandard color (2 mg/kg or less).
Loss on drying: 9.0 % or less (1 g, 105 °C, 4 hours)
Assay: 0.25 g of this product is weighed to the digits of 0.001 g and the value is recorded. It
is added with 40 mL of water, subjected to ultrasonic vibration for 1 minute, transferred
to a 50 mL volumetric flask, added with water to the graduation line to make 50 mL. 5.0
mL of this solution measured using a volumetric pipette is transferred to a 50 mL
volumetric flask and added with water to the graduation line to make 50 mL. This is used
as a sample solution. 20 µL of this solution is tested by the liquid chromatography under
the following conditions. The peak height or area of L-ascorbic acid-2-phosphate ester ismeasured from the obtained chromatogram. The concentration of L-ascorbic acid-2-

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phosphate ester is obtained from a calibration curve separately prepared. Then, its content
is calculated using the following equation.
Amount of sodium -calcium -L-ascorbic acidate-2-phosphate ester (%)=C×

50 319.10
×W 553.63

C: Concentration of L-ascorbic acid-2-phosphate ester (mg/mL)
W: Amount of this product weighed (g)Operating condition:
Detector: Ultraviolet spectrophotometer (measurement wavelength: 250 nm)
Column: A stainless tube (inner diameter: 4.6 mm, length: 150 mm) is filled with particle
size 5 µm of octadecyl-silylated silica gel for liquid chromatography.
Column temperature: Constant temperature around 25 °C
Mobile phase: 13.6 g (13.55~13.64 g) of potassium dihydrogenphosphate and 4.0 mL oftetrabutylammonium hydroxide test solution are dissolved in 950 mL of water, the pHof the solution is adjusted to 6.0 using 2 mol/L sodium hydroxide test solution, andthen water is added to make 1,000 mL. 950 mL of this solution is added with 50 mL ofacetonitrile to mix them.
Flow rate: The flow rate is adjusted so that the retention time of L-ascorbic acid-2phosphate ester is approximately 5 minutes.
Preparation of calibration curve:
Approximately 0.1 g of the reference standard of L-ascorbic acid-2-phosphate ester tris
cyclohexylammonium is weighed to the digits of 0.001 g and the value is recorded. It isdissolved with water, transferred to a 20 mL volumetric flask, added with water to thegraduation line to make 20 mL. This is used as a standard stock solution. 1 mL of thissolution contains 5 mg of L-ascorbic acid-2-phosphate ester tris cyclohexylammonium(C24H48N3O9P). In use, a certain volume of the standard stock solution is diluted with water
so that 1 mL of it contains 0.2 mg, 0.4 mg, 0.6 mg or 0.8 mg. This is used as a standard
solution. 20 µL each of these standard solutions is hereinafter tested by the liquid
chromatography as the same is the case in the sample solution. The peak height or area of Lascorbic acid-2-phosphate ester is measured from the obtained chromatogram and thecalibration curve is prepared.
(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation(a) Compositional standards

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Provisional Translation from Japanese Original

The compositional standards of the raw material for manufacturing of sodium-calciumL-ascorbic acid-2-phosphate ester are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodiumcalcium-L-ascorbic acid-2-phosphate ester is applied mutatis mutandis.

(22) L-Ascorbic acid-2-Phosphate ester magnesium
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 93.0 % or more of L-ascorbic acid-2phosphate ester magnesium ((C6H6O9P)2Mg3·10H2O).
Physical and chemical properties:
i. This product is white to slightly yellow powder with a slight, specific odor.
ii. This product is easy to dissolve in water and hardly dissolves in acetone or methanol.
iii. The pH of the solution of this product in water (1 → 50) is 7.5~8.5.
Confirmation test:i. 2 mg (1.5~2.4 mg) of this product is added with 0.1 mol/L of hydrochloric acid test
solution to make 100 mL. In the measurement of the absorption spectrum of thisproduct, the absorption maximum is at the wavelength of 235~239 nm.
ii. When 5 mL of a solution of this product in water (1 → 50) is added with a drop of
ferric chloride test solution, the resulting solution is red-brown.
iii. 0.1 g (0.05~0.14 g) of this product is added with 2 mL of sulfuric acid and 15 mL ofhydrogen peroxide solution, heated until it is approximately 5 mL, cooled, and then is
added with water to make 50 mL. When 1 mL of this solution is added with 1 mL of 3
mol/L of sulfuric acid, 1 mL of ammonium molybdate test solution, and 1 mL ofamidol-sodium bisulfite test solution, the resulting solution turns blue.
iv. 0.5 g (0.45~0.54 g) of this product is added with 2 mL of sulfuric acid and 30 mL ofhydrogen peroxide solution and heated until it is approximately 5 mL. When it allowed
to cool, added with 20 mL of water, and neutralized with 1 mol/L sodium hydroxide
test solution, the resulting solution gives the qualitative reaction of magnesium salt.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is dissolved
with 10 mL of water, the solution shall not be darker than the control solution for colorJ, and should be almost clear.

272

Provisional Translation from Japanese Originalii. Specific rotation: Approximately 1.0 g of this product is weighed to the digits of 0.01
g and the value is recorded. It is dissolved in water to make 50 mL. The rotation of thissolution shall be [α]20
D = +44° ~ +50°.
iii. Chloride: 0.05 g (0.045~0.054 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.5 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.35 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 0.2 g (0.15~0.24 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Water content: Approximately 1.6 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is placed to a 50 mL volumetric flask, dissolved with a mixture ofmethanol and sulfuric acid (70:1), added with more mixture of methanol and sulfuric acid(70:1) to the graduation line to make 50 mL. This is used as a sample solution. The massof the solution is weighed to three significant digits and the value is recorded. 1 mL ofthis sample solution is measured using a volumetric pipette and water content isdetermined by the direct titration method, the Karl Fischer method. Separately, the massof approximately 1 mL of a mixture of methanol and sulfuric acid (70:1) is weighed tothree significant digits and the value is recorded. The water content of this mixture isdetermined in the same way. The water content of this product determined using thefollowing equation shall be 22.0~28.0 %.
Water content of this product (%) =50 × S - B × ( w2 - w1 ) / w3
w1 × 10

S: Water content in 1 mL of the test solution (mg)
B: Water content in approximately 1 mL of a mixture of methanol and sulfuric acid(70:1), the mass of which is weighed to three significant digits and the value isrecorded (mg)
W1: Amount of this product weighed (g)W2: Mass of 50 mL of the sample solution (g)273

Provisional Translation from Japanese Original

W3: Mass of approximately 1 mL of a mixture of methanol and sulfuric acid (70:1)
used for the measurement of B (g)Assay: Approximately 0.3 g each of this product and approximately 0.50 g of maleic acid as
an internal standard is weighed to the digits of 0.001 g and the value is recorded. They are
dissolved with water to make 100 mL to prepare a sample solution. 20 µL of this solution
is tested by the liquid chromatography under the following conditions. The peak areas SA1
and SM1 of L-ascorbic acid-2-phosphate ester magnesium and maleic acid, respectively,
are measured from the obtained chromatogram. Separately, the peak areas SA2 and SM2 ofthe reference standard of L-ascorbic acid-2-phosphate ester magnesium and maleic acid,respectively, are measured by the same procedure.
Content of L-ascorbic acid-2-phosphate ester magnesium ((C6H6O9P)2Mg3·10H2O) (%) =(SA1/WA1) × (WM1/SM1) × (WA2/WM2) × (SM2/SA2) × 100
WA1: Amount of this product weighed (g)WM1: Collected amount of maleic acid related to this product (g)WA2: Collected amount of the reference standard of L-ascorbic acid-2-phosphate ester
magnesium (g)WM2: Collected amount of maleic acid related to the reference standard of L-ascorbic
acid-2-phosphate ester magnesium (g)Operating condition:
Detector: Ultraviolet spectrophotometer (measurement wavelength: 257 nm)
Column: A stainless tube (inner diameter: 8 mm, length: 250 mm) is filled with the gel ofpolyvinyl alcohol cross-linked with epichlorohydrin, as a packing material (exclusion
limit molecular weight (pullulan) is 1.8 × 103).
Column temperature: 40 °C
Mobile phase: A solution for which 7.10 g (7.095~7.104 g) of anhydrous sodium sulfate
and 5.76 g (5.755~5.764 g) are dissolved in water to make 1,000 mL.
Flow rate: 0.7 mL per minute(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-ascorbic acid-2phosphate ester magnesium are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-ascorbic acid2-phosphate ester magnesium is applied mutatis mutandis.

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Provisional Translation from Japanese Original(23) Astaxanthin
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 97.0~102.0 % of astaxanthin(C40H52O4).
Physical and chemical properties: This product is reddish violet to dark purple, crystalline
powder with a slight, specific odor.
Confirmation test:
i. When 0.5 mL of a solution of this product in chloroform (1 → 10,000) is added with 1
mL of antimony trichloride test solution, the resulting solution is bluish purple within
30 seconds.
ii. In the measurement of the absorption spectrum of the sample solution obtained by theassay using chloroform as a control, the absorption maximum is at the wavelengths of
487~491 nm.
Purity test:
i. Clarity and color of solution: When 0.01 g (0.005~0.014 g) of this product is dissolved
with 10 mL of chloroform, the resulting solution shall be red and clear.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (0.5 g, 105 °C, 4 hours)
Ignition residue: 0.10 % or less (0.5 g)
Assay: Approximately 0.035 g of this product is weighed to the digits of 0.1 mg and thevalue is recorded. It is dissolved with chloroform, transferred to a 200 mL volumetricflask, and added with more chloroform to the graduation line to make 200 mL. 5 mL ofthis solution is measured using a volumetric pipette and put in a 50 mL volumetric flask
and added with chloroform to the graduation line to make 50 mL. Then, 5 mL of thissolution is measured using a volumetric pipette and put in a 50 mL volumetric flask andadded with chloroform to the graduation line to make 50 mL. This is used as a sample

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solution. The absorbance A of this solution is measured at a wavelength of nearly 489 nm
using chloroform as a control.
Amount of astaxanthin (C40H52O4) (mg) =A× 200,000
1,975(b) Standard of storage method
It shall be put in a lightproof hermetically sealed container with inert gas replaced with
air and stored.
B. Preparation (Part 1)(a) Compositional standards
This product is particles of the mixture of the raw material for manufacturing of
Astaxanthin and carrier.
Content: When this product is determined, it contains Astaxanthin (C40H52O4)
corresponding to 90~120 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 mg of
the raw material for manufacturing of astaxanthin is weighed, added with 1 mL ofwarmed water at 60 °C, and sonicated in a water bath at 60 °C for approximately 5
minutes. It is allowed to cool, added with 10 mL of chloroform and shaken up well. 1
mL of the chloroform layer is collected and added with 1 mL of antimony trichloride
test solution. The resulting solution is bluish-purple within 30 seconds.
ii. In the measurement of the absorption spectrum of the sample solution obtained by theassay using chloroform as a control, the absorption maximum is at the wavelengths of
485~489 nm.
iii. 20 mL of the supernatant obtained by the assay is collected and the solvent is distilledaway under reduced pressure. The residue is added with 0.5 mL of chloroform and 10µL of this solution is spotted on a thin layer plate prepared using silica gel for thinlayer chromatography. Then it is developed approximately 15 cm with the developing
solvent, a mixture of cyclohexane and dioxane (3:2) in a dark place. When the thinlayer is air dried in a dark place, red spots are observed at the Rf value ofapproximately 0.4.
Assay: The amount of this product containing approximately 5 mg of astaxanthin(C40H52O4) is weighed to three significant digits and the value is recorded. It is addedwith approximately 5 mL of warmed water at 60 °C, and sonicated in a water bath at
60 °C for approximately 5 minutes until the particles disappear. It is allowed to cool,transferred to a 200 mL volumetric flask, added with 100 mL of absolute ethanol, shaken
up well, then added with chloroform to the graduation line to make 200 mL.

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Approximately 40 mL of this solution is measured and centrifuged for 5 minutes at 3,000rpm. 5 mL of the supernatant is measured using a volumetric pipette and the solvent isdistilled away in a water bath at 50 °C under nitrogen stream. The residue is dissolved
with chloroform by 5-minute sonication, transferred to a 50 mL volumetric flask andadded with chloroform to the graduation line to make 50 mL. This is used as a sample
solution. The absorbance A of this solution is measured at a wavelength of nearly 487 nm
using chloroform as a control.
Amount of astaxanthin (C40H52O4) (mg) =A× 200,000
1,830(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
C. Preparation (Part 2: liquid)(a) Compositional standards
This product is suspension of the mixture of the preparation (Part 1), in which the raw
material for manufacturing of astaxanthin is mixed with edible starch, mixed with vegetableoil, light anhydrous silicic acid, glycerine fatty acid ester or polyoxyethylene sorbitan fatty
acid ester.
Content: When this product is determined, it contains astaxanthin (C40H52O4) corresponding
to 90~120 % of the amount on the label.
Confirmation test: The confirmation test of the preparation (Part 1) of astaxanthin is appliedmutatis mutandis.
Assay: The assay of the preparation (Part 1) of astaxanthin is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the preparation (Part 1) of astaxanthin is appliedmutatis mutandis.
(c) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
Valid period: 3 months from the day of the manufacture“有効期間 製造の日から３か月”(24) Acetomenaphthone
A. Raw material for manufacturing(a) Compositional standards

277

Provisional Translation from Japanese OriginalContent: When this product is determined following 3-hour drying at 80 °C, it contains
98.0 % or more of astaxanthin (C15H14O4).
Physical and chemical properties: This product is white crystalline powder with no odor or aslight acetic acid odor.
Confirmation test:
i. 0.03 g (0.025~0.034 g) of this product is dissolved with 2 mL of glacial acetic acid,added with 2 mL of dilute hydrochloric acid, and heated in a water bath for 5 minutes.
It is allowed to cool, added with a drop of strong hydrogen peroxide solution, warmed
for a while, shaken up, added with 2 mL of water and 3 mL of chloroform, and shaken
up, and then the chloroform layer is yellow.
ii. 1 mL of the chloroform layer of i. is measured and warmed to evaporate chloroform.
When the residue is dissolved with 1 mL of ethanol, added with 1 mL of ammonia test
solution, shaken up, and added with 3 drops of ethyl cyanoacetate, the resulting
solution is purple-blue. When 1 mL of sodium hydroxide solution (1 → 3) is added tothis solution, the solution turns to green, and then to brownish.
iii. 0.2 g (0.15~0.24 g) of this product is added with 3 mL of 1 mol/L sodium hydroxide
test solution, heated for several minutes, allowed to cool, and neutralized with dilute
sulfuric acid. The generated precipitation is filtered. The filtrate gives the qualitative
reaction of acetate.
Purity test:
i. Melting point: The melting point of this product shall be 112~115 °C.
ii. Zinc: 1.0 g (0.95~1.04 g) of this product is weighed, added with 10 mL of dilutehydrochloric acid, heated until acetomenaphthone becomes oily, cooled within 30
seconds, and filtered. The filter paper is washed with 30 mL of boiling water. Whenthe mixture of the filtrate and the washings are added with water to make 50 mL,
added with 1.0 mL of potassium ferrocyanide test solution, shaken up, and allowed tostand for 5 minutes, the turbidity of this solution shall be lower than or equal to thatshown when 5.0 mL of zinc reference standard is added with 10 mL of dilutehydrochloric acid and subjected hereinafter to the same procedure for the sample
solution (50 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, 80 °C, 3 hours)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried at 80 °C for 3 hours, approximately 0.25 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is placed in a 150 mL saponification
flask, dissolved with 20 mL of glacial acetic acid, added with 15 mL of dilutehydrochloric acid, boiled for 15 minutes with a reflux condenser, and water-cooled.

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Within 30 seconds after the water-cooling, it is titrated with 0.1 mol/L ceric ammonium
sulfate solution (indicator: 3 drops of o-phenanthroline test solution). A blank test is
performed in the same way and corrections are made.
0.1 mol/L ceric ammonium sulfate solution 1 mL = 12.91 mg C15H14O4(b) Standard of storage method
It shall be stored in a lightproof tightly sealed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of
acetomenaphthone are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of
acetomenaphthone is applied mutatis mutandis.

(25) β-apo-8'-carotene acid ethyl ester
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 96.0~102.5 % of β-apo-8'-carotenneacid ethyl ester (C32 H44O2).
Physical and chemical properties:
i. This product is red-brown to reddish violet crystalline powder.
ii. This product is easy to dissolve in chloroform, is hard to dissolve in ether, hard to
extremely dissolve in cyclohexane, and hardly dissolves in absolute ethanol and water.
iii. This product gradually changes by oxygen and light.
iv. Melting point: About 132 °C (decomposition)
Confirmation test:i. 1 mg (0.5~1.4 mg) of this product is dissolved with 10 mL of chloroform. When 0.5
mL of this solution is measured and added with 1 mL of antimony trichloride test
solution, the resulting solution is green.
ii. In the measurement of the absorption spectrum of the sample solution obtained by theassay, the absorption maximum is at the wavelengths of 446~450 nm and 471~475 nm
and the absorption minimum is at 461~467 nm. When the absorbances at the former
and latter absorption maximum are A1 and A2, respectively, A1/A2 is 0.82~0.86.

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Provisional Translation from Japanese Original

Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, reduced pressure, silica gel, 3 hours)
Ignition residue: 0.10 % or less (1 g)Assay: Approximately 10 mg of this product is weighed to the digits of 0.1 mg and thevalue is recorded. It is dissolved with cyclohexane, transferred to a 200 mL volumetricflask and added with more cyclohexane to the graduation line to make 200 mL. 4 mL ofthis solution is measured using a volumetric flask, and transferred to a 100 mL volumetricflask, and added with cyclohexane to the graduation line to make 100 mL. This is used asa sample solution. The absorbance A of this solution is measured at a wavelength ofnearly 448 nm using cyclohexane as a control.
Amount of β-apo-8'-carotene acid ethyl ester (C32H44O2) (mg) =(b) Standard of storage method

A× 50,000
2,550Store in airtight container shielded from light, replace air with nitrogen gas and save.
B. Preparation(a) Component standard
This product is particles of the mixture of the raw material for manufacturing of β-apo-8'carotenic acid ethyl ester and fillers.
Content: When this product is determined, it contains β-apo-8'-carotenic acid ethyl ester(C32H44O2) corresponding to 90~120 % of the amount on the label.
Confirmation test: 5 mL of the supernatant obtained by the assay is measured and thesolvent is distilled away in a water bath at 50 °C under nitrogen stream. The residue isadded with 0.5 mL of chloroform and 10 µL of this solution is spotted on a thin layer
plate prepared using silica gel for thin-layer chromatography. Then it is developed
approximately 15 cm with the developing solvent, a mixture of dichloromethane andether (9:1) in a dark place. When the thin layer is air dried in a dark place, red spots areobserved at the Rf value of approximately 0.7.

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Assay: The amount of this product containing approximately 10 mg of β-apo-8'-carotene
acid ethyl ester (C32H44O2) is weighed to three significant digits and the value is recorded.
It is added with 10 mL of warmed water at 60 °C, warmed in a water bath at 60 °C,
stirred under nitrogen stream to make it a complete suspension. It is allowed to cool andtransferred to a 250 mL volumetric flask, added with 50 mL of absolute ethanol and 100
mL of ether, vigorously shaken up for 3 minutes and added with ether to the graduationline to make 250 mL. Then, it is allowed to stand in a dark place for 15 minutes toprecipitate insoluble matter and the 5 mL of the supernatant is measured using a
volumetric pipette, and placed in a 100 mL a round-bottom flask, and the solvent isdistilled away in a water bath at 50 °C under nitrogen stream. After cooling, the residue isdissolved with cyclohexane and added with more cyclohexane to the graduation line to
make 100 mL. This is used as a sample solution. The absorbance A of this solution ismeasured at a wavelength of nearly 449 nm using cyclohexane as a control.
Amount of β-apo-8'-carotene acid ethyl ester (C32H44O2) (mg) =(b) Standard of storage method

2,430

× 50,000

Store in an airtight container shielded from light.
(26) Aminoacetic acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.5 % or more of aminoacetic acid (C2H5NO2).
Physical and chemical properties:
i. This product is white crystals or crystalline powder with a sweet taste.
ii. This product is soluble in water and hardly soluble in ethanol or ether.
iii. The pH of the aqueous solution (1 → 10) of this product is 5.5~7.0.
Confirmation test:
i. When 5 mL of the solution of this product in water (1 → 10) is added with 5 drops ofdilute hydrochloric acid and 1 mL of sodium nitrite test solution, colorless gas is
generated.
ii. 5 drops of the solution, which completes the reaction of i., is placed in a small test
tube, boiled for a while, evaporated to dryness in a desiccator at 120 °C, and allowed
to cool. When the residue is added with 5~6 drops of chromotropic acid test solution
and heated in a water bath for 10 minutes, the solution is dark purple.

281

Provisional Translation from Japanese Originaliii. When 5 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes, the solution is purple.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) is dissolved with 10 mL ofwater, the solution shall be colorless and clear.
ii. Chloride: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by the chloride test method. A control solution is prepared using 0.30 mL of 0.01
mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.021 %).
iii. Ammonium salt: 0.1 g (0.05~0.14 g) of this product is placed in a distilling flask,
dissolved with 70 mL of water, added with 1 g (0.5~1.4 g) of magnesium oxide, anddistilled until the volume of distillate liquid reaches 40 mL. A receiver is a Nessler
tube containing 2 mL of 0.1 mol/L hydrochloric acid, in which the bottom of thecondenser is immersed. When the distillate liquid is added with 5 mL of 1 mol/L
sodium hydroxide test solution and water to make 50 mL and added with 1 mL of
Nessler reagent solution, the color shall not be darker than that obtained when 2 mL ofammonium reference standard is placed in a Nessler tube, added with water to make
40 mL, and hereinafter subjected to the same procedure as one for the sample (0.02 %or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.3 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 0.1 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours, approximately 0.5 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolves in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (indicator: 0.5 mL of α-naphtholbenzein test solution). In this case,282

Provisional Translation from Japanese Original

the end point of the titration is the point at which the color of the solution changes frombrown to green. A blank test is performed in the same way and corrections are made.
01.mol/L perchloric acid 1 mL = 7.507 mg C2H5NO2(b)Standard of strong method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The Compositional standards of raw material for manufacturing of aminoacetic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of aminoacetic
acid is applied mutatis mutandis.

(27) DL-alanine
A. Raw material for manufacturing(a) Compositional standard
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.5 % or more of DL-alanine (C3H 7NO2).
Physical and chemical properties:
i. This product is colorless to white crystalline powder with a sweet taste.
ii. This product is easy to dissolve in water, it is difficult to dissolve in organic solvents.
iii. The pH of the aqueous solution (1 → 10) of this product is 5.5~7.0.
Confirmation test:
i. When 0.2 g (0.15~0.24 g) of this product is dissolved in 10 mL of dilute sulfuric acid,added with 0.1 g (0.05~0.14 g), and boiled, it emits the odor of acetaldehyde.
ii. When 5 ml of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes, it is purple.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is dissolved in
10 mL of water, the solution shall be colorless and clear.
ii.

Chloride: When chloride is tested using a sample solution prepared with 0.5 g(0.45~0.54 g) of this product by the chloride test method and a control solution
prepared with 0.3 mL of 0.01 mol/L hydrochloric acid, the turbidity of the sample
solution shall not be higher than that of the control solution (0.021 % or less).

283

Provisional Translation from Japanese Originaliii. Ammonium salt: The purity test iii. of the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis, provided that “2 mL of ammonium
reference standard” shall be replaced by “3 mL of ammonium reference standard”(0.03 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less). v. Arsenic: 1.0 g (0.95~1.04 g) of this product isweighed and a sample solution is prepared by Method No. 1 of the arsenic test method.
The arsenic test is performed by the method using device A. The color of absorbing
solution shall not be darker than the standard color (2 mg/kg or less).
Loss on drying: 0.3 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 0.20 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours, approximately 0.2 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (indicator: 10 drops of α-naphtholbenzein test solution). In thiscase, the end point of the titration is the point at which the color of the solution changes
from brown to green. A blank test is performed in the same way and corrections aremade.
01.mol/L perchloric acid 1 mL = 8.909 mg C3H7NO2(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of DL-alanine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of DL-alanine isapplied mutatis mutandis.

(28) L-arginine
A. Raw material for manufacturing(a) Compositional standards284

Provisional Translation from Japanese OriginalContent: When this product is determined after dried at 105 °C for 3 hours, it contains
98.0 % or more of L-arginine (C6H14N4O2).
Physical and chemical properties:
i. This product is pale yellow to pale yellow-brown crystals or crystalline powder with aslight, specific odor.
ii. The pH of a solution or a suspension of this product in water (1 → 10) is 10.5~12.0.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes, the solution is reddish violet topurple.
ii. When 2 mL of a solution of this product in water (1 → 20,000) is added with 1 mL of
8-oxyquinoline test solution and 1 mL of N-bromosuccinimide solution (1 → 1,000),
the solution is red-yellow.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours, approximately 4 g of it isweighed to the digits of 0.01 g, and the value is recorded. It is dissolved in 6 mol/Lhydrochloric acid to make 50 mL and filtered using a membrane filter (0.45 µm). The
rotation of this solution shall be[α]20
D = + 25.5 to + 29.5°.

ii. Ammonium salt: The purity test iii. of the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis, provided that “0.1 g” and “2 mL ofammonium reference standard” shall be replaced by “0.02 g” and “3 mL of ammonium
reference standard”, respectively (0.15 % or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed in a decomposition flask,
added with 10 mL of nitric acid and 5 mL of sulfuric acid, and gently heated. If thesolution is still brown, it is allowed to cool, then is heated with 1~2 mL of additional
nitric acid. This procedure is repeated until the solution becomes colorless to slightly
yellow. It is allowed to cool, added with 0.5 mL of perchloric acid, and heated until
white smoke emerges. It is allowed to cool, added with 15 mL of saturated ammonium
oxalate solution and again heated until white smoke emerges. It is allowed to cool andadded with water to make approximately 10 mL. This is used as a sample solution.

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When the sample solution is tested on arsenic by the method using device A, the colorof absorbing solution shall not be darker than the standard color (2 mg/kg or less).
Loss on drying: 1.0 % or less (1 g, 105 °C., 3 hours)
Ignition residue: 1.0 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours, approximately 0.2 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved with 0.5 mL of water and 50
mL of glacial acetic acid, and titrated with 0.1 mol/L perchloric acid (indicator: 1 mL of
α-naphtholbenzein test solution). Provided, however, that the end point of the titration is
the point at which the color of the solution changes from orange-yellow throughyellowish green to green. A blank test is performed in the same way and corrections are
made.
0.1 mol/L perchloric acid 1 mL = 8.710 mg C6H14N4O2(b) Standard of storage method

Store in a light-tight closed container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-arginine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-arginine isapplied mutatis mutandis.

(29) L- Isoleucine
A. Raw material for manufacturing(a) Compositional standards
Content: L-isoleucine, after drying at 105 °C for four hours, contains not less than 90.0% of
L-isoleucine (C6H13NO2).
Physical and chemical properties:
i. L-isoleucine occurs as white crystals or crystalline powder.
ii. The pH of an aqueous solution of L-isoleucine or a suspension of L-isoleucine in water(0.2 → 20) is between 4.5 ~ 7.0.
Identification: To 5 mL of an aqueous solution of L-isoleucine (1 → 1000), add 1 mL ofninhydrin solution (1 → 1000), and heat for three minutes: a purple color develops.

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Provisional Translation from Japanese Original

Purityi. Specific rotation: Weigh about 2 g of L-isoleucine, previously dried at 105 °C for fourhours, to the order of 0.01 g, record the value, dissolve in 6 mol/L hydrochloric acid TS tomake 50 mL, filter if necessary, and determine the optical rotation of this solution: It shallbe between +38.0 ~ +41.5°.
ii. Ammonium salt: Proceed as directed in the Purity (3) for the active ingredients foraminoacetic acid production. In this case, the term “0.1 g” shall be deemed to be replacedwith “0.01 g,” and the term “2 mL of Standard Ammonium Solution” shall be deemed tobe replaced with “3 mL of Standard Ammonium Solution” (not more than 0.30%).
iii. Lead: Perform the test for lead with 0.5 g (0.45 ~ 0.54 g) of L-isoleucine as directedunder Lead Test (Method 1 of Atomic Absorption Spectrophotometry): the amount oflead shall be not more than 2 μg/g. Pipet 0.5 mL of standard lead solution using a
volumetric pipette, transfer it into a 50-mL volumetric flask, add nitric acid (1 → 150)
to the marked line to make 50 mL, and use this solution as the standard solution.
iv. Arsenic: Weigh 1.0 g (0.95 ~1.04 g) of L-isoleucine, transfer to a decomposition flask,
add 10 mL of nitric acid and 5 mL of sulfuric acid, and heat gently. If a brown color stilldevelops in the solution, allow to cool, add 1 to 2 mL of nitric acid, heat, and repeat thisprocedure until the solution becomes colorless to pale yellow. After allowing to cool, add
0.5 mL of perchloric acid, and heat until white fumes are evolved. After allowing to cool,
add 15 mL of saturated ammonium oxalate solution, and heat again until white fumes are
evolved. After allowing to cool, add water to make about 10 mL, and use this solution as
the sample solution to perform the test for arsenic by using Apparatus A . At this time,
the color of the absorbing liquid shall have no more color than the standard color (notmore than 2 μg/g).
Loss on drying: Not more than 2.0% (5 g, 105°C, 4 hours)
Residue on ignition: Not more than 1.0% (1 g)Assay: Weigh about 0.25 g of L-isoleucine, previously dried at 105 °C for four hours, tofour significant figures, record the value, dissolve it in water, transfer to a 250-mL
volumetric flask, add water to the marked line to make 250 mL, and use this solution as
the sample solution. Perform the test with 5 μL of this sample solution as directed under
Liquid Chromatography according to the following conditions. Based on thechromatogram obtained, determine the peak area of L-isoleucine, calculate theconcentration of L-isoleucine by a calibration curve obtained separately, and calculate thecontent.

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Provisional Translation from Japanese Original

Operating conditions
Apparatus: The apparatus consists of two pumps for flowing the mobile phase and thechromogenic liquid, a sample introduction part, a column, a reaction vessel, a detector,
and a recording apparatus, and use the column and the reaction vessel that can be kept at aconstant temperature.
Detector: A fluorometer (excitation wavelength: 338 nm, fluorescence wavelength: 425 nm)
Column: A stainless steel column 4.6 mm in inside diameter and 150 mm in length, packed
with octadecylsilanized silica gel for liquid chromatography (3 μm in particle diameter)
Column temperature: 60 °C
Reaction vessel (reaction coil): A column about 0.25 mm in inside diameter and about 3.0 m
in length
Mobile phase: Dissolve 3.4 g of potassium dihydrogen phosphate and 1.62 g of sodium 1octanesulfonate in water to make 1000 mL. To this solution, add 266 mL of methanol, andadjust the pH to 2.5 with phosphoric acid.
Reaction solution: Dissolve 13.25 g of potassium hydroxide, 15.00 g of boric acid, 0.35 g ofo-phthalaldehyde, 1 mL of 2-mercaptoethanol, 5 mL of methanol, and 1.25 mL of 3.5%
polyoxyethylene (23) lauryl ether in water to make 1000 mL.
Mobile phase flow rate: 1.0 mL per minute
Reaction solution flow rate: 0.5 mL per minute
Reaction temperature: 60 °C
Creating calibration curve
Weigh about 0.25 g of L-isoleucine for assay to the order of 0.001 g, record the value, add
water to dissolve it, transfer to a 50 mL volumetric flask, add water to the marked line to
make 50 mL, and use this solution as the standard stock solution [each mL of this solution
contains 5 mg of L-isoleucine (C6H13NO2)]. When using it, dilute a fixed volume of thestandard stock solution with water exactly so that each mL contains 0.5 mg, 1.0 mg, and
1.5 mg. Filter each solution through a 0.45 μm membrane filter as necessary, and use thesesolutions as the standard solutions. Perform the test with 5 μL each of the standard
solutions as directed under Liquid Chromatography in the same manner as for the sample
solution. Based on the chromatogram obtained, determine the peak area of L-isoleucine,and prepare a calibration curve.
(b)Standards for manufacturing method
Cultivate Corynebacterium glutamicum strain KCCM 80189, and after the completion ofculturing, filter the culture to remove bacterial bodies and isolate the L-isoleucine crude
crystalline fraction. Purify a crude crystal and dry the resulting solid to produce theproduct.

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Provisional Translation from Japanese Original(c) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-isoleucine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-isoleucine isapplied mutatis mutandis.

(30) Inositol
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following 4hours drying at 105 °C, it contains
97.0 % or more of inositol (C6H12O6).
Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor and with a sweet
taste.
ii. This product is easy to dissolve in water and hardly dissolves in ether, ethanol, andchloroform.
iii. A solution of this product in water (1 → 10) is neutral.
iv. This product is not optical rotatory.
Confirmation test:i. 1 mL of a solution of this product in water (1 → 50) is added with 6 mL of nitric acidand evaporated to dryness on a water bath. When the residue is added with 0.5 mL ofstrontium nitrate solution (1 → 10) and again evaporated to dryness in a water bath, itchanges through reddish violet to orange.
ii. When 4 mL of a solution of this product in water (1 → 100) is added with 1 mL oflead subacetate test solution, shaken up, and heated in a water bath for 5 minutes, thesolution becomes a translucent gel.
iii. The melting point of hexa-acetyl-inositol obtained by the assay is 212~216 °C.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) is dissolved with 10 mL ofwater, the resulting solution shall be colorless and clear.
ii. Melting point: The melting point of this product shall be 223~227 °C.

289

Provisional Translation from Japanese Originaliii. Chloride: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample solution
by the chloride test method. When sulfate is tested using a control solution preparedwith 0.30 mL of 0.01 mol/L hydrochloric acid using the chloride test method, theturbidity of the sample solution shall not be higher than that of the control solution(0.005 % or less).
iv. Sulfate: 4.0 g (3.95~4.04 g) of this product is weighed to prepare a sample solution by
the sulfate test method. When sulfate is tested using a control solution prepared with
0.50 mL of 0.005 mol/L sulfuric acid using the sulfate test method, the turbidity of thesample solution shall not be higher than that of the control solution (0.006 % or less).
v. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.5 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (25 mg/kg or less).
vi. Iron: When 2.0 g (1.95~2.04 g) of this product is weighed, dissolved with 40 mL ofwater, and added with 2 mL of hydrochloric acid, 0.04 g (0.035~0.044 g) ofammonium persulfate and 2 mL of ammonium thiocyanate test solution, the color of
the resulting solution shall not be darker than that obtained when 1.0 mL of ironreference standard is measured, added with 40 mL of water, and hereinafter subjectedto the same procedure as the one for the sample (5 mg/kg or less).
vii. Calcium: When 1.0 g (0.95~1.04 g) of this product is dissolved with 10 mL of water,
added with 1 mL of ammonium oxalate test solution, and allowed to stand for 1
minute, the resulting solution shall be clear.
viii. Fehling test solution reducing substance: When 0.5 g (0.45~0.54 g) of this product isdissolved with 10 mL of water, added with 5 mL of Fehling test solution, boiled for 3
minutes, and allowed to stand for 30 minutes, orange-yellow to red precipitation shallnot be generated.
Loss on drying: 0.5 % or less (1 g, 105 °C, 4 hours)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried at 105 °C for 4 hours, and approximately 0.2 g of it isweighed to the digits of 0.001 g, the value is recorded. It is placed in a 250 mL beaker
and added with 5 mL of a mixture of 1 mL of dilute sulfuric acid and 50 mL of acetic
anhydride. The beaker is covered with a watch glass, heated on a water bath for 20
minutes, and cooled with ice. Then it is added with 100 mL of water, boiled for 20
minutes and allowed to cool. The contents of the beaker are placed in a 250 mL
separatory funnel, and then washed with a little water. Then the beaker is washed290

Provisional Translation from Japanese Original

sequentially with 30, 25, 20, 15, 10, and 10 mL of chloroform and extracted
sequentially. All chloroform extracts are collected together and washed with 10 mL ofwater and the chloroform layer is filtered with absorbent cotton. The aqueous layer andthe absorbent cotton are washed with 10 mL of chloroform. The mixture of the filtrate
and the washings are evaporated to dryness on a water bath, dried at 105 °C for 2 hours,
allowed to cool, and the mass is weighed. The mass is the amount of hexa-acetylinositol (C18H24O12).
Amount of inositol (C6H12O6) (mg) =Amount of hexa-acetyl-inositol (C18H24O12) (mg) × 0.4167(b) Standard of storage method

It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of inositol are appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of inositol isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of inositol
and fillers.
Content: When this product is determined, it contains inositol (C6H12O6) corresponding to
90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 g of the
raw material for manufacturing of inositol is weighed, added with 50 mL of water,
shaken up well, and filtered. To the filtrate, the confirmation test i. for the raw material
for manufacturing of inositol is hereinafter applied mutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 1 g of the
raw material for manufacturing of inositol is weighed, added with 100 mL of water,
shaken up well, and filtered. To the filtrate, the confirmation test ii. for the raw
material for manufacturing of inositol is hereinafter applied mutatis mutandis.
iii. The melting point of hexa-acetyl-inositol obtained by the assay is 212~216 °C.
Assay: According to the amount of this product on the label, the amount containing
approximately 1.0 g of inositol (C6H12O6) is weighed to three significant digits and the291

Provisional Translation from Japanese Originalvalue is recorded. It is added with 60 mL of water, shaken up well, and filtered. The
residue on the filter is washed twice with 15 mL of water. The filtrate and the washings
are collected together, transferred to a 100 mL volumetric flask, added with water to thegraduation line to make 100 mL. 20 mL of this solution is measured using a volumetricpipette, placed in a 250 mL beaker, evaporated to dryness on a water bath, and dried at
105 °C for an hour. It is allowed to cool, and the assay for the raw material for
manufacturing of inositol is hereinafter applied mutatis mutandis.
Amount of inositol (C6H12O6) (mg)
= Amount of hexa-acetyl-inositol (C18H24O12) (mg) × 0.4167(b) Standard of storage method

The standard of storage method of the raw material for manufacturing of inositol isapplied mutatis mutandis.

(31) Ergocalciferol
A. Raw material for manufacturing(a) Compositional standards
Physical and chemical properties:
i. This product is white crystals with no odor.
ii. This product is easy to dissolve in ethanol, acetone, ether or chloroform, easy toslightly dissolve in fat and oil, and hardly dissolves in water.
iii. This product changes by air or light.
Confirmation test:
i. When 0.5 mg (0.45~0.54 mg) is dissolved with 5 mL of chloroform, added with 0.3
mL of acetic anhydride and 0.1 mL of sulfuric acid, and shaken up, the resulting
solution is red and within 30 seconds changes from purple through blue to green.
ii. 0.05 g (0.045~0.054 g) of this product is dissolved with 1 mL of anhydrous pyridine,
added with the solution of 0.05 g (0.045~0.054 g) of 3,5-dinitrobenzoyl chloride in 1
mL of anhydrous pyridine, and heated on a water bath for 10 minutes with a reflux
glass tube. It is allowed to cool and added with 5 mL of water, and the generated
precipitation is filtered, washed with water, recrystallized twice with acetone as asolvent, and dried in a desiccator (reduced pressure, silica gel) for 2 hours, and thenthe melting point is 147~149 °C.

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Provisional Translation from Japanese Original

Purity test:
i. Absorbance: 0.01 g of this product is weighed to the digits of 0.0001 g and the value isrecorded. It is dissolved in ethanol to make 1,000 mL. The absorbance of this solution
measured at wavelength of 265 nm, shall be E11 %cm = 445~485.
ii. Specific rotation: 0.3 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved in ethanol within 30 minutes to make 20 mL. The rotationof this solution measured within 30 minutes shall be [α]20
D = +102~+107° .

iii. Melting point: This product is placed in a capillary and dried in a desiccator (reducedpressure, 2.67 kPa or less) for 3 hours. When the capillary is sealed by melting within
30 seconds and heated in a bath heated at a temperature, which is approximately 10 °C
less than the predicted melting point, at the rate of 3 °C increase per 1 minute, themelting point shall be 115~118 °C.
iv. Ergosterol: When 0.010 g (0.0095~0.0104 g) of this product is dissolved with 2.0 mLof absolute ethanol (9 → 10), added with a solution of 0.020 g (0.0195~0.0204 g) ofdigitonin in 2.0 mL of absolute ethanol (9 → 10), and allowed to stand for 18 hours,
precipitation shall not be generated.
(b) Standard of storage method
It shall be put in a lightproof sealed container with nitrogen replaced with air and stored
in a cold place.
B. Preparation (Part 1: liquid)(a) Compositional standards
This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of ergocalciferol and hydrogenated oils, higher saturated fatty acids, fatty
acids, vegetable oil, or animal fats.
Content: When this product is determined, it contains ergocalciferol (C28H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing vitamin D
powder are applied mutatis mutandis.
Assay: The test is performed using the vitamin D assay.
(b) Standard of storage method
It shall be put in a lightproof air tight container making it almost full or with nitrogen
replaced with air.
C. Preparation (Part 2: powder)(a) Compositional standards
It is powder or particles, in which the raw material for manufacturing of ergocalciferol
and fillers are mixed.

293

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains ergocalciferol (C28H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing vitamin D
powder are applied mutatis mutandis.
Assay: The test is performed using the vitamin D assay.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

(32) Potassium Chloride
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following 2 hours drying at 130 °C, it contains
99.0 % or more of potassium chloride (KCl).
Physical and chemical properties:
i. This product is colorless or white crystals or crystalline powder with no odor and witha salty taste.
ii. This product is easy to dissolve in water, and hardly dissolves in ethanol.
iii. Solution of this product in water (1 → 50) is neutral.
Confirmation test: A solution of this product in water (1 → 50) gives the qualitative
reactions of potassium salt and chloride.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is dissolved
with 5 mL of water, the solution shall be colorless and clear.
ii. Acid and alkali: When 5.0 g (4.95~5.04 g) of this product is weighed, dissolved with
50 mL of newly boiled and cooled water, added with 3 drops of phenolphthalein test
solution, it shall not be red. When this solution is added with 0.50 mL of 0.01 mol/L
sodium hydroxide solution, the resulting solution shall be red.
iii. Bromide: 1.0 g (0.95~1.04 g) of this product is weighed and dissolved with water tomake 100 mL. When 5 mL of this solution is measured, added with 3 drops of dilutehydrochloric acid and 1 mL of chloroform and 3 drops of chloramine test solution are
dropped in it with shaking, the chloroform layer shall not be yellow to yellow-red.
iv. Iodide: When 0.5 g (0.45~0.54 g) of this product is weighed, dissolved with 10 mL ofwater, added with 3 drops of ferric chloride test solution and 1 mL of chloroform,
shaken up, allowed to stand for 30 minutes, and again shaken up, the chloroform layer
shall not be reddish violet to purple.

294

Provisional Translation from Japanese Originalv. Heavy metal: 4.0 g (3.95~4.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (5 mg/kg or less).
vi. Calcium and magnesium: When 0.20 g (0.195~0.204 g) of this product is weighed,dissolved with 20 mL of water, added with 2 mL of ammonia test solution, 2 mL ofammonium oxalate test solution, and 2 mL of disodium hydrogen phosphate test
solution, and allowed to stand for 5 minutes, the solution shall not have opacity.
vii. Sodium: When 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with 20 mL
of water, and subjected to the flame coloration test, it shall not be continuously yellow.
viii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, 130 °C, 2 hours)
Assay: This product is dried at 130 °C for 2 hours, approximately 0.2 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved with 50 mL of water andtitrated with 0.1 mol/L silver nitrate solution (indicator: 1 mL of potassium chromate test
solution) with vigorous shaking. A blank test is performed in the same way andcorrections are made.
0.1 mol/L silver nitrate solution 1 mL = 7.455 mg KCl(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of potassium chloride
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
chloride is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of potassium
chloride and fillers.

295

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains potassium chloride (KCl)corresponding to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of potassium chloride is weighed,added with 50 mL of water, shaken up well, and filtered. It is neutralized with dilutenitric acid as appropriate, and to this solution the confirmation test for the raw material
for manufacturing of potassium chloride is applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.2 g of potassium chloride(KCl) is weighed to three significant digits, and the value is recorded. It is added with 20
mL of water, shaken up well, and filtered. The residue on the filter is washed six times
with 5 mL of water, the filtrate and the washings are collected together and neutralizedwith dilute nitric acid as appropriate. The assay for the raw material for manufacturing ofpotassium chloride is hereinafter applied mutatis mutandis.
0.1 mol/L silver nitrate solution 1 mL = 7.455 mg KCl(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
chloride is applied mutatis mutandis.

(33) Choline Chloride
A. Raw material for manufacturing(a) Compositional standards
Content: By the determination, this product contains 96.0 % or more of choline chloride(C5H14ClNO) on the dried basis.
Physical and chemical properties:
i. This is colorless, almost clear, and viscous fluid with a slight, specific odor.
ii. This product is miscible with ethanol and hardly dissolves in ether, chloroform, or
benzene.
iii. The pH of this product is 6.5~8.0.
iv. This product is hygroscopic and absorbs carbon dioxide to emit an amine odor.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 100) is added with 3 mL ofreinecke salt test solution, red precipitation is generated.
ii. When 5 mL of a solution of this product in water (1 → 10) is added with 2 g (1.5~2.4g) of potassium hydroxide and warmed, it emits an amine odor and its gas changes wet
red litmus paper to blue.

296

Provisional Translation from Japanese Originaliii. A solution of this product in water (1 → 10) gives the qualitative reaction of chloride.
Purity test:
i. Ethylene glycol: 10.0 g (9.95~10.04 g) of this product is weighed into a stoppered
flask, added with 25 mL of periodic acid test solution, allowed to stand in a dark placefor 30 minutes, added with 20 mL of potassium iodide test solution, shaken up, andtitrated with 0.1 mol/L sodium thiosulfate solution (indicator: 1 mL of starch test
solution). A blank test is performed in the same way and corrections are made. The
amount of ethylene glycol shall be 1.0 % or less.
0.1 mol/L sodium thiosulfate solution 1 mL = 6.207 mg C2H6O2ii. Trimethylamine: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with water,transferred to a 100 mL volumetric flask, added with more water to the graduation line
to make 100 mL. This is used as a sample solution. 5 mL of the sample solution ismeasured, transferred to a separatory funnel, added with 1 mL formalin-magnesium
carbonate test solution, 10 mL of toluene and 3 mL of saturated potassium carbonate,vigorously shaken up for 1 minute, and allowed to stand for 5 minutes. The toluene
layer is transferred to a stoppered test tube containing 0.5 g (0.45~0.54 g) of anhydrous
sodium sulfate and toluene is dehydrated. When 5 mL of this toluene solution is addedwith 5 mL of picric acid-toluene test solution, the color of the resulting solution shallnot be darker than that of a solution prepared with 5 mL of trimethylamine reference
standard solution by the same procedure for the preparation of the sample solution.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 30 % or less (0.5 g, reduced pressure, phosphorus pentoxide, 5 hours)
Ignition residue: 0.20 % or less (1 g)Assay: Approximately 0.3 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is completely dissolved with 50 mL of mercuric acetate test solution
fornonaqueous titration, added with 10 mL of mercuric acetate test solution for
nonaqueoustitration and titrated with 0.1 mol/L perchloric acid (indicator: 2 drops ofmethylrosanilinium chloride test solution). In this case, the end point of the titration is thepoint at which the color of the solution changes from purple through blue to green. A
blank test is performed in the same way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 13.96 mg C5H14ClNO

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Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in an airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of choline chloride
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of choline chloride
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particle of the mixture of the raw material for manufacturingof choline chloride and fillers.
Content: When this product is determined, it contains choline chloride (C5H14ClNO)
corresponding to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amount of thisproduct containing 1 g of the raw material for manufacturing of choline chloride isweighed, added with water to make 100 mL, shaken up well, and filtered. To the filtrate,
the confirmation test i. for the raw material for manufacturing choline chloride ishereinafter applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.3 g of choline chloride(C5H14ClNO) is measured to three significant digits and the value is recorded. It is addedwith 50 mL of methanol and sufficiently eluted for 30 minutes with occasional shakingup, filtered, and washed with 20, 15, and 15 ml of methanol. The filtrate and thewashings are collected together and evaporated to dryness on a water bath. It is addedwith 50 mL of glacial acetic acid for nonaqueous titration and warmed on a water bath tocompletely dissolve. The assay for the raw material for manufacturing of choline chloride
is hereinafter applied mutatis mutandis.
0.1 mol/L perchloric acid 1 mL = 13.96 mg C5H14ClNO(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of choline chloride
is applied mutatis mutandis.

298

Provisional Translation from Japanese Original(34) Dibenzoyl Thiamine Hydrochloride
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (reduced pressure, silica gel) for 24hours and determined, it contains 97.0 % or more of dibenzoyl thiamine hydrochloride(C26H26N4O4SꞏHCl).
Physical and chemical properties:
i. This product is white crystalline powder with no odor.
ii. This product is easy to dissolve in chloroform, hard to dissolve in ethanol or acetone,
hard to extremely dissolve in water, and hardly dissolves in ether.
Confirmation test:
i. When 0.1 g (0.05~0.14 g) of this product is dissolved with 10 mL of methanol, addedwith 1 mL of dilute nitric acid, and then added with 1 mL of silver nitrate test solution,
white a precipitation is generated.
ii. 5 mg (4.5~5.4 mg) of this product is added with 1 mL of methanol, warmed todissolve, added with 2 mL of water, 2 mL of cysteine hydrochloride test solution (1→ 100) and 2 mL of pH 7.0 phosphate buffer, shaken up, and allowed to stand for 30
minutes. Then it is added with 1 mL of potassium ferricyanide test solution, 5 mL of
0.5 mol/L sodium hydroxide test solution and 5 mL of isobutanol, vigorously shaken
up for 2 minutes, allowed to stand to separate the solution into two layers, then is
irradiated with ultraviolet rays from above. When the top of the upper layer isobserved from a perpendicular direction to the direction of the irradiation, bluishpurple fluorescence is observed. This fluorescence disappears when the solution ismade acidic, and reappears when it is made alkaline.
iii. 30 mg (29.5~30.4 mg) of this product is added with 7 mL of 0.1 mol/L hydrochloric
acid test solution, heated in a water bath to dissolve, added with 2 mL of a mixture ofhydroxylamine hydrochlorides solution (3 → 20) and sodium hydroxide solution(3 → 20) (1:1), shaken up for a minute, and added with 0.8 mL of hydrochloric acidand 0.5 mL of ferric chloride test solution. The resulting solution is purple.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is added with 10 mLof water and heated in a water bath to dissolve. The resulting solution shall be clear or
almost clear.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using

299

Provisional Translation from Japanese Original

these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 11.0 % or less (1 g, reduced pressure, silica gel, 5 hours)
Ignition residue: 0.20 % or less (1 g)Assay: This product is dried in a desiccator (reduced pressure, silica gel) for 24 hours,
approximately 0.4 g of it is weighed to the digits of 0.001 g, and the value is recorded. It
is dissolved with 40 mL of methanol and 40 mL of 0.1 mol/L hydrochloric acidtestsolution, transferred to a 1,000 mL volumetric flask and added with water to thegraduation line to make 1,000 mL. This is used as a sample solution. 5 mL of thissolution is measured using a volumetric pipette, transferred to a 250 mL volumetric flask,
and added with 0.1 mol/L hydrochloric acid test solution to the graduation line to make
250 mL. The absorbance A of this solution is measured at a wavelength of 237 nm.
Separately, a blank test is performed in the same way and the absorbance A0 is measured.
Amount of dibenzoyl thiamine hydrochloride (C26H26N4O4SꞏHCl) (mg) =A - A0×500,000
526(b) Standard of storage method
It shall be stored in an airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of dibenzoyl thiaminehydrochloride are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of dibenzoyl
thiamine hydrochloride is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particle of the mixture of the raw material for manufacturing ofdibenzoy thiamine hydrochloride and fillers.
Content: When this product is determined, it contains dibenzoyl thiamine hydrochloride(C26H26N4O4SꞏHCl) corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.025 g of
the raw material for manufacturing of dibenzoyl thiamine hydrochloride is weighed,added with 15 mL of a mixture of 0.1 mol/L hydrochloric acid test solution andethanol (3:7), vigorously shaken up for 1 minute, and filtered. 5 mL of the filtrate isadded with 2 mL of a mixture of hydroxylamine hydrochlorides solution (3 → 20) and300

Provisional Translation from Japanese Original

sodium hydroxide solution (3 → 20) (1:1), shaken up for 1 minute, added with 0.8 mLof hydrochloric acid and 0.5 mL of mercuric chloride test solution, and filtered. The
resulting solution is purple.
ii. 0.3 mL of the filtrate in i.

is added with 2 mL of cysteine hydrochloride test solution(1 → 100) and 2 mL of pH 7.0 phosphate buffer, shaken up, and allowed to stand for
30 minutes. Then, it is added with 3 mL of cyanogen bromide test solution fordibenzoyl thiamine for assay, 5 mL of 1 mol/L sodium hydroxide test solution and 5
mL of isobutanol, vigorously shaken up for 2 minutes, allowed to stand to separate thesolution into two layers. When it is irradiated with ultraviolet rays from above and the
top of the upper layer is observed from a perpendicular direction to the direction of theirradiation, bluish~purple fluorescence is observed. This fluorescence disappears when
the solution is made acidic, and reappears when it is made alkaline.
Assay: The amount of this product containing approximately 0.03 g of dibenzoyl thiaminehydrochloride (C26H26N4O4S·HCl) is weighed to three significant digits and the value isrecorded. It is added with 200 mL of a mixture of 0.1 mol/L hydrochloric acid test
solution and ethanol (3:7) using a volumetric pipette, is vigorously shaken up for 5
minutes, and filtered. 2 mL of the filtrate is measured using a volumetric pipette,
transferred to a 100 mL volumetric flask, added with 5 mL of cysteine hydrochloride
solution (1 → 20), added with dilute sodium hydroxide test solution to make it pH
7.0~7.5, and added with water to the graduation line to make 100 mL. This solution isshaken up well and allowed to stand for 60 minutes to prepare a sample solution.
Separately, the reference standard of thiamine hydrochloride is dried at 105 °C for 2
hours, approximately 0.02 g of it is weighed to the digits of 0.0001 g and the value isrecorded. It is added with 200 mL of a mixture of 0.1 mol/L hydrochloric acid test
solution and ethanol (3:7) using a volumetric pipette to dissolve. 2 mL of this solution ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, addedwith 5 mL of cysteine hydrochloride solution (1 → 20), added with dilute sodiumhydroxide test solution to make it pH 7.0~7.5, and added with water to the graduationline to make 100 mL. This is used as a standard solution. 2 mL of the sample solution ismeasured using a volumetric pipette into stoppered centrifugal precipitation tubes T and
T’, added with 3 mL of acidic potassium chloride test solution, then added with 1 g(0.5~1.4 g) of anhydrous sodium sulfate, warmed in a water bath at 50~60 °C for 20
minutes with occasional, gentle shaking-up to make it almost dissolve. It is cooled toroom temperature. The stoppered centrifugal precipitation tube T is added with 3 mL of
cyanogen bromide test solution for dibenzoyl thiamine assay, shaken up, added with 2
mL of sodium hydroxide solution (3 → 10) within 30 seconds, shaken up, added with 15

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mL of isobutanol using a volumetric pipette, tightly stoppered, and shaken up for 2
minutes. The stoppered centrifugal precipitation tube T’ is added with 2 mL of sodiumhydroxide solution (3isobutanol using a volumetric pipette, tightly stoppered, and shaken
updibenzoyl thiamine assay, shaken up, added with 15 mL of isobutanol using a
volumetric pipette, tightly stoppered, and vigorously shaken up for 2 minutes. Separately,
2 mL of the standard solution is measured using a volumetric pipette into stoppered
centrifugal precipitation tubes S and S’, and subjected to the same procedure for the
sample solution. Each centrifugal precipitation tube is centrifuged and each isobutanol
layer is collected. Fluorescence intensities FT, FT’, FS, and FS’ of the collected layers aremeasured at the maximum wavelength of excitation at around 370 nm and at the
maximum wavelength of fluorescence at around 440 nm using a fluorometer.
Amount of dibenzoyl thiamine hydrochloride (C26H26N4O4SꞏHCl) (mg)
= Amount of reference standard of thiamine hydrochloride (mg)×(b) Standard of storage method

FT - FT'
×1.5627
FS - FS'

It shall be stored in a capped container.

(35) Thiamine Hydrochloride A. Raw material for manufacturing
A. Raw material for manufacturing(a) Compositional standards
Content: This product contains 98.0 ~102.0 % of thiamine hydrochloride(C12H17ClN4OS·HCl) when you determine the quantity of it after drying for 2 hours at
105 degree.
Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor or a slight, specificodor.
ii. This product is easy to dissolve in water, hard to dissolve in ethanol or glycerin, andhardly dissolves in ether or benzene.
iii. Melting point: About 245 °C (decomposition)
Confirmation test:
i. When 2 mL of a solution of this product in water (1 → 500) is added with 2~3 drops ofiodine test solution, with 2~3 drops of Mayer's test solution, and with 1 mL of picricacidtest solution, red-brown precipitation or opacity, yellowish white precipitation or
opacity and yellow precipitation or opacity are generated, respectively.

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Provisional Translation from Japanese Originalii. When 1 mL of a solution of this product in water (1 → 500) is added with 1 mL oflead acetate test solution and 1 mL of sodium hydroxide solution (1 → 10) andwarmed, the solution turns through yellow to brown. It is allowed to stand and thenblackish brown precipitation is generated.
iii. When 5 mL of a solution of this product in water (1 → 500) is added with 2.5 mL of 1
mol/L sodium hydroxide test solution and 0.5 mL of potassium ferricyanide test
solution, then added with 5 mL of isobutanol, vigorously shaken up for 2 minutes,
allowed to stand, and observed under ultraviolet, the isobutanol layer emits blue-violet
fluorescence. This fluorescence disappears when the solution is made acidic, andreappears when it is made alkaline.
iv. A solution of this product in water (1 → 500) gives the qualitative reaction ofchloride.
Purity test:
i. pH: 1.0 g (0.95~1.04 g) of this product is dissolved in water to make 100 mL. The pHof this solution shall be 2.7~3.4.
ii. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved in water
to make 10 mL. The color of this solution shall not be darker than that of a solution
prepared by which 1.5 mL of 0.017 mol/L potassium dichromate solution is addedwith water to make 1,000 mL.
iii. Sulfate: 1.5 g (1.45~1.54 g) of this product is weighed to prepare a sample solution by
the sulfate test method. A control solution is prepared using 0.35 mL of 0.005 mol/L
sulfuric acid. When sulfate is tested using these samples and control solutions, theturbidity of the sample solution shall not be higher than that of the control solution(0.011 % or less).
iv. Nitrate: When 0.5 g (0.45~0.54 g) of this product is dissolved with 25 mL of water. 2
mL of this solution is added with 2 mL of sulfuric acid, shaken up, and allowed tocool, and on it the layer of ferrous sulfate test solution is formed, at the boundary
surface a dark brown ring shall not be produced.
v. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 5.0 % or less (0.5 g, 105 °C, 2 hours)
Ignition residue: 0.20 % or less (1 g)

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Provisional Translation from Japanese Original

Assay: This product and the reference standard of thiamine hydrochloride are separately
dried at 105 °C for 2 hours. Approximately 0.1 g each is weighed to the digits of 0.001 g
and the value is recorded. Each of them is dissolved with 0.001 mol/L hydrochloric acidtest solution, transferred to a 200 mL volumetric flask, and added with 0.001 mol/Lhydrochloric acid test solution to the graduation line to make 200 mL. 2 mL each of thesesolutions is measured using a volumetric pipette and transferred to a 50 mL volumetricflask, added with 0.001 mol/L hydrochloric acid test to the graduation line to make 50
mL. They are used as a sample solution and a standard solution. 5 mL of the sample
solution is measured using a volumetric pipette into a stoppered test tube T and T’. The
tube T is added with 3.0 mL of cyanogen bromide, shaken up, added with 5.0 mL of
sodium hydroxide solution (1 → 10) within 30 seconds and shaken up. The tube T’ isadded with 5.0 mL of sodium hydroxide solution (1 → 10), shaken up, added with 3.0
mL of cyanogen bromide and shaken up.Separately, 5 mL of the standard solution ismeasured using a volumetric pipette into stoppered test tubes S and S’, and the sameprocedure for the sample solution is performed.Absorbance of each solution, AT, AT’, AS,
AS’ at a wavelength 368 nm measured using water as a control solution.
Amount of thiamine hydrochloride (C12H17ClN4OSꞏHCl) (mg)
= Amount of reference standard of thiamine hydrochloride (mg) ×

AT - AT'AS - AS'(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of thiaminehydrochloride are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of thiaminehydrochloride is applied mutatis mutandis.

(36) L- Histidine hydrochloride
A. Raw material for manufacturing(a) Compositional standards
Content: L-histidine hydrochloride, after drying at 105 °C for three hours, contains not lessthan 98.0% of L-histidine hydrochloride monohydrate (C6H9N3O2.HCl.H2O).

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Provisional Translation from Japanese Original

Physical and chemical properties

i. L-histidine Monohydrochloride occurs as white crystals or crystalline powder.
ii. The pH of an aqueous solution of L-histidine hydrochloride (1 → 10) is between 3.5 ~4.5.
Identificationi. To 5 mL of an aqueous solution of L-histidine hydrochloride (1 → 1000), add 1 mL ofninhydrin solution (1 → 1000), and heat for three minutes: a purple color develops.
ii. An aqueous solution of L-histidine hydrochloride (1 → 10) responds to the Qualitative
Tests for chloride.
Purityi. Specific rotation: Weigh about 5.5 g of L-histidine hydrochloride to the order of 0.01 g,
record the value calculated on the dried basis, dissolve in 6 mol/L hydrochloric acid TS tomake 50 mL, filter if necessary, and determine the optical rotation of this solution: It shallbe between + 8.5 ~ + 10.5°.

ii. Ammonium salt: Proceed as directed in the Purity (3) for the active ingredients foraminoacetic acid production. In this case, the term “0.1 g” shall be deemed to be replacedwith “0.05 g,” and the term “1 mL” shall be deemed to be replaced with “0.5 mL” (notmore than 0.04 %).

iii. Lead: Perform the test for lead with 5.0 g (4.95 ~ 5.04 g) of L-histidine hydrochloride
as directed under Lead Test (Method 1 of Atomic Absorption Spectrophotometry): The
amount of lead shall be not more than 2 μg/g.
Pipet 1.0 mL of standard lead solution using a volumetric pipette, transfer it into a 10 mL
volumetric flask, add nitric acid (1 →150) to the marked line to make 10 mL, and use thissolution as the standard solution.
iv. Arsenic: Transfer 1.0 g (0.95 ~ 1.04 g) of L-histidine hydrochloride to a decomposition
flask, add 10 mL of nitric acid and 5 mL of sulfuric acid, and heat gently. If a brown color
still develops in the solution, allow to cool, add 1 to 2 mL of nitric acid, heat, and repeat
this procedure until the solution becomes colorless to pale yellow. After allowing to cool,
add 0.5 mL of perchloric acid, and heat until white fumes are evolved. After allowing tocool, add 15 mL of saturated ammonium oxalate solution, and heat again until white fumes
are evolved. After allowing to cool, add water to make about 10 mL, and use this solution
as the sample solution to perform the test for arsenic by using Apparatus A .
At this time, the color of the absorbing liquid shall have no more color than the standard
color (not more than 2 μg/g).
Loss on drying: Not more than 0.3% (3 g, 105 °C, 3 hours)
Residue on ignition: Not more than 0.1% (1 g)

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Provisional Translation from Japanese Original

Assay: Weigh about 0.5 g of L-histidine hydrochloride, previously dried at 105 °C for threehours, to the order of 0.1 mg, record the value, add water to dissolve it, transfer to a 1000mL volumetric flask, add water to the marked line to make 1000 mL, and use this solution
as the sample solution. Perform the test with 5 μL of this solution as directed under liquid
chromatography according to the following conditions. Based on the chromatogram
obtained, determine the peak area of histidine, and calculate the concentration of L-histidinehydrochloride monohydrate by a calibration curve obtained separately, and calculate thecontent.
Operating conditions
Detector: An ultraviolet absorption photometer (measuring wavelength: 210 nm)
Column: A stainless steel column 4.6 mm in inside diameter and 150 mm in length packed
with octadecylsilanized silica gel for liquid chromatography (3 μm in particle diameter).
Column temperature: 35 °C
Mobile phase: Dissolve 2.2 g (2.265 ~ 2.274 g) of potassium dihydrogen phosphate and 1.08
g (1.075 ~ 1.084 g) of sodium 1-octanesulfonate in 850 mL of water, adjust the pH to 2.5
with phosphoric acid, add and mix 100 mL of acetonitrile for liquid chromatography, andadd water to make 1000 mL.
Flow rate: 1.0 mL per minute
Creating calibration curve
Weigh about 0.05 g, 0.1 g, 0.5 g, and 1 g of L-histidine hydrochloride for assay to the order of
0.1 mg, record the values, add about 800 mL of water to each to dissolve it, transfer to1000 mL volumetric flasks, add water to the marked line to make 1000 mL, and use thesesolutions as the standard solutions containing 0.05 mg, 0.1 mg, 0.5 mg, and 1 mg per mL.
Perform the test with 5 μL each of the standard solutions as directed under Liquid
Chromatography in the same manner as for the sample solution. Based on thechromatogram obtained, determine the peak area of histidine and prepare a calibration
curve.
(b) Standard of storage method
Culture a histidine-producing strain of Corynebacterium glutamicum aerobically, and after
the completion of culturing, filter the culture to remove bacterial bodies and isolate the Lhistidine crude crystalline fraction. Purify a crude crystal and dry the resulting solid to
produce the product.
(c) Standard of storage method
It shall be stored in a capped container.
B. Preparatio(a) Compositional standards

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Provisional Translation from Japanese Original

The compositional standards of the raw material for manufacturing of L-histidinehydrochloride are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-histidinehydrochloride is applied mutatis mutandis.

(37) Pyridoxine Hydrochloride
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 4 hours drying in a desiccator(reduced pressure, silica gel), it contains 98.0% or more of pyridoxine hydorochloride(C8H11NO3·HCl).
Physical and chemical properties:
i. This product is white-slightly yellow crystalline powder with no odor and with bitterand sour tastes.
ii. This product is easy to dissolve in water, hard to dissolve in ethanol, and hardly
dissolves in acetone, ether or chloroform.
iii. The pH of solution (1 → 50) of this product is 2.5~3.5.
iv. This product gradually changes by light.
v. Melting point: About 206 °C (decomposition)
Confirmation test:
i. When 1 mL of a solution of this product in water (1 → 1,000) is added with a drop of
ferric chloride test solution, the resulting solution is orange-brown. The color changes
to yellow by adding a drop of hydrochloric acid.
ii. When 1 mL of solution of this product in water (1 → 10,000) is added with 2 mL of
newly prepared a solution of 2,6-dibromequinonechloroimide in ethanol (1 → 4,000)
and a drop of ammonia test solution, the resulting solution is blue. When 1 mL of asolution of this product in water (1 → 10,000) is added with 1 mL of a saturated
solution of boric acid and subjected to the same procedure, the solution is not blue.
iii. 0.5 g (0.45~0.54 g) of this product is added with 1 mL of water, warmed to dissolve,
allowed to cool, added with 6 mL of picric acid test solution, and allowed to stand for
2~3 hours. The deposited crystals are collected by filtration, washed with a little icewater, dried at 105 °C for 2 hours, and the melting point is 156~159 °C(decomposition).
iv. A solution of this product in water (1 → 10) gives the qualitative reaction of chloride.

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Provisional Translation from Japanese Original

Purity test: Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 3.0 mL of lead standard
solution, the color of the sample solution shall not be darker than that of the control
solution (30 mg/kg or less).
Loss on drying: 0.30 % or less (1 g, reduced pressure, silica gel, 4 hours)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried in a desiccator (reduced pressure, silica gel) for 4 hours.
Approximately 0.4 g of it is weighed to the digits of 0.001 g and the value is recorded. It
is added with 10 mL of mercuric acetate test solution for nonaqueous titration, shaken up
to dissolve, added with 60 mL of glacial acetic acid for nonaqueous titration and titrated
with 0.1 mol/L perchloric acid (indicator: 2 drops of methylrosanilinium chloride test
solution). In this case, the end point of the titration is the point at which the color of thesolution changes from purple to blue. A blank test is performed in the same way andcorrections are made.
0.1 mol/L perchloric acid 1 mL = 20.56 mg C8H11NO3・HCl(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Pyridoxinehydrochloride are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Pyridoxinehydrochloride is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof pyridoxine hydorochloride and fillers.
Content: When this product is determined, it contains pyridoxine hydorochloride(C8H11NO3·HCl) corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.01 g of
the raw material for manufacturing of pyridoxine hydrochloride is weighed, addedwith 10 mL of water, and for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fat added with 3 mL of308

Provisional Translation from Japanese Originalchloroform, shaken up, and allowed to stand, and the aqueous layer is filtered toprepare a sample solution. 1 mL of this solution is measured, and hereinafter theconfirmation tests i. for the raw material for manufacturing of pyridoxinehydrochloride is applied mutatis mutandis.
ii. 1 mL of the sample solution i. is measured and added with water to make 10 mL. 1 mLof this solution is measured, and hereinafter the confirmation tests ii. for the raw
material for manufacturing of pyridoxine hydrochloride is applied mutatis mutandis.
iii. When 1 mL of the sample solution i. is measured and added with 0.5 mL ofphosphotungstic acid test solution, the resulting solution becomes cloudy.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forothers.
Method No. 1: The amount of this product containing approximately 0.02 g of pyridoxinehydrochloride (C8H11NO3・HCl) is weighed to three significant digits and the value isrecorded. It is added with 50 mL of chloroform, shaken up, and filtered using a glass
filter (G4). The residue on the filter is washed three times with 20 mL of chloroform.
When the chloroform odor on the residue disappears, the residue is filtered byextracting with approximately 90 mL of warm water, allowed to cool, transferred to a
100 mL volumetric flask. The filtrate is added with water to the graduation line to
make 100 mL. 25 mL of this solution is measured using a volumetric pipette into a 200mL volumetric flask and added with water to the graduation line to make 200 mL. Thisis used as a sample solution. Separately, the reference standard of pyridoxinehydrochloride is dried in a desiccator (reduced pressure, silica gel) for 4 hours.
Approximately 0.1 g of it is weighed to the digits of 0.001 g and the value is recorded.
It is dissolved with water, transferred to a 100 mL volumetric flask and added withmore water to make 100 mL. 5 mL of this solution is measured using a volumetricpipette, transferred to a 200 mL volumetric flask, added with water to the graduationline to make 200 mL. This is used as a standard solution. 1 mL of the sample solution
is measured using a volumetric pipette, placed in a 25 mL volumetric flask, added with
2.0 mL of barbital buffer, 9.0 mL of isopropanol and 2.0 mL of newly prepared
solution of 2,6-dibromequinonechloroimide in ethanol (1 → 4,000), shaken up well,added with isopropanol to the graduation line to make 25 mL, and allowed to stand for
90 minutes. For the resulting solution, the absorbance AT at the maximum wavelength
at around a wavelength of 650 nm is measured. The solution prepared using 1 mL ofwater instead of the sample solution by the same procedure for the sample solution isused as a control solution. Separately, 1 mL of the standard solution is measured using

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Provisional Translation from Japanese Original

a volumetric pipette. Its absorbance AS is measured by the same procedure for the
sample solution.
Amount of pyridoxine hydrochloride (C8H11NO3・HCl) (mg)
= Amount of reference standard of pyridoxine hydrochloride (mg) ×

AT 1
×
AS 5

Method No. 2: The amount of this product containing approximately 0.02 g of pyridoxinehydrochloride (C8H11NO3・HCl) is weighed to three significant digits and the value isrecorded. It is added with 60 mL of water, vigorously shaken up, and filtered and theresidue is washed twice with 10 mL of water. The filtrate and the washings arecollected together, transferred to a 100 mL volumetric flask, added with water to thegraduation line to make 100 mL. 25 mL of this solution is measured using a
volumetric pipette, transferred to a 200 mL volumetric flask, added with water to thegraduation line to make 200 mL. This solution is used as a sample solution andhereinafter the Method No. 1 is applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container..

(38) L-Lysine Monohydrochloride
L-lysine monohydrochloride (Part 1)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.5 % or more of L-lysine monohydrochloride (C6H14N2O2·HCl).
Physical and chemical properties:
i. This product is white to light brown powder with no odor or a slight, specific odor.
ii. This product is easy to dissolve in water and extremely hard to dissolve in ethanol.
iii. The pH of a solution (1 → 10) of this product is 5.0~6.0.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution, heated for 3 minutes, added with 20 mL of water, and allowed
to stand for 15 minutes, the resulting solution is reddish violet.
ii. A solution of this product in water (1 → 10) gives the qualitative reaction of chloride.
iii. 0.1 g (0.05~0.14 g) each of this product and Japanese pharmacopeia lysinehydrochloride is weighed and dissolved with 10 mL of water. They are used as sample
and standard solutions. These solutions are tested by the thin layer chromatography. 5310

Provisional Translation from Japanese OriginalµL each of the sample and standard solutions is spotted on a thin layer plate preparedusing silica gel for thin-layer chromatography. Then it is developed approximately 10
cm with the developing solvent, a mixture of n-propanol and strong ammonia solution(67:33) and the thin layer is air dried. When it is sprayed with a solution of ninhydrin
in acetone (1 → 50) and dried at 80 °C for 5 minutes, the spots obtained from thesample and standard solutions are purple and their Rf values are equal.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours. Approximately 4 g of it isweighed to the digits of 0.01 g and the value is recorded. It is dissolved in hydrochloric
acid (13 → 25) to make 50 mL and as appropriate filtered using a membrane filter(0.45 µm). The rotation of this solution measured at a layer length 100 mm shall be[α]20
D = +18.0 ~ +21.5°.

ii. Ammonium salt: The purity test iii. for the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis. In this case, “0.1 g” and “1 mL” shall be
replaced by “0.05 g” and “0.5 mL”, respectively (0.04 % or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 3.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (30 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 1.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 0.3 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours. Approximately 0.2 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is added with 10 mL of mercuric acetate
test solution for nonaqueous titration, warmed to dissolve, allowed to cool, added with 50
mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1 mol/L perchloric
acid (indicator: 10 drops of α-naphtholbenzein test solution). In this case, the end point of
the titration is the point at which the color of the solution changes from orange to yellowgreen. A blank test is performed in the same way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 9.133 mg C6H14N2O2∙HCl(b) Standard of storage method
It shall be stored in a capped container.

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Provisional Translation from Japanese Original

B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-lysine
monohydrochloride (part 1) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-lysine
monohydrochloride (part 1) is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof L-lysine monohydrochloride (part 1) and excipient.
Content: When this product is determined, it contains L-lysine monohydrochloride(C6H14N2O2·HCl) corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.1 g ofraw material for manufacturing of L-lysine monohydrochloride (Part 1) is weighed,added with 100 mL of water, shaken up and filtered. 5 mL of the filtrate is added with
1 mL of ninhydrin test solution, hereinafter the confirmation test i. of the raw material
for manufacturing of L-lysine monohydrochloride (Part 1) is applied mutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 0.1 g ofraw material for manufacturing of L-lysine monohydrochloride (Part 1) is weighed,added with 10 mL of water, shaken up and filtered. The filtrate is used as a sample
solution. Separately, 0.1 g (0.05~0.14 g) of lysine hydrochloride is weighed and addedwith 10 mL of water to prepare a standard solution. The confirmation test iii. of the
raw material for manufacturing of L-lysine monohydrochloride (Part 1) is hereinafter
applied mutatis mutandis.
Assay: The amount of this product containing approximately 1.0 g of L-lysine
monohydrochloride (C6H14N2O2∙HCl) is weighed to three significant digits and the value
is recorded. It is dissolved with water, placed in a 500 mL volumetric flask, added withwater to the graduation line, shaken up well to make 500 mL, and filtered using a dried
filter paper. 20 mL of the first filtrate is removed, and 20 mL of the next filtrate ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, addedwith water to the graduation line to make 100 mL. This is used as a sample solution.
Separately, lysine hydrochloride is dried at 105 °C for 3 hours. Approximately 0.05 g and0.03 g of it are weighed to the digits of 0.1 mg and the values are recorded. Each of themis dissolved with water, transferred to a 100 mL volumetric flask, added with water to the312Provisional Translation from Japanese Original

graduation line to make 100 mL. The two solutions are used as standard solutions S and
S'. 2 mL each of the sample solution and the standard solutions S and S' is measured
using a volumetric pipette, transferred to a 50 mL volumetric flask, added with 4 mL ofninhydrin test solution for assay using a volumetric pipette, heated in a water bath for 20
minutes, allowed to cool, and then added with water to the graduation line to make 50
mL. The absorbances AT, AS and AS at the maximum wavelength at around wavelength
475 nm are measured for the sample solution and the standard solutions S and S',
respectively.
Amount of L-lysine monohydrochloride (C6H14N2O2∙HCl) (mg)A- A

= b + AT - AS'SS'× (a - b)

a: Standard S: Amount of L-lysine monohydrochloride in 1 mL (mg)b: Standard S': Amount of L-lysine monohydrochloride in 1 mL (mg)(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-lysine
monohydrochloride (part 1) is applied mutatis mutandis.
L-Lysine Monohydrochloride (Part 2)
A. Raw material for manufacturing(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-lysine
monohydrochloride (part 1) are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the L-lysine producing recombinant, whose host is the strain
belonging to Escherichia coli, is cultured. After the cultivation, the bacterial cells are heattreated to separate L-lysine, and dried.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-lysine
monohydrochloride (part 1) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-lysine
monohydrochloride (part 2) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-lysine
monohydrochloride (part 2) is applied mutatis mutandis.

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Provisional Translation from Japanese Original(39) L-Carnitine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.0~102.0 % of L-carnitine(C7H15NO3) at dehydration product conversion.
Physical and chemical properties:
i. This product is white powder.
ii. The pH of a solution of this product in water (1 → 20) is 6.5 to 8.5.
Confirmation test: When the infrared absorption spectrum of this product is measured using
the potassium bromide disk method of the infrared absorption spectroscopy, main
absorptions are observed at wavenumbers around 3,460 cm−1, 1,600 cm−1, 1,479 cm−1,
1,393 cm−1, and 945 cm−1.
Purity test:i. Specific rotation: 10 g of this product is weighed to the digit of 0.01 g and the value isrecorded. It is dissolved with water to make 100 mL. The optical rotation of thissolution shall be [α]20
D = -30.0 ~ -32.0°.

ii. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 10
mL of water. The resulting solution shall be colorless and clear.
iii. Chloride: 5.0 g (4.95~5.04 g) of this product is weighed to prepare a sample solution
using the chloride test method. A control solution is prepared using 2.80 mL of 0.01
mol/L hydrochloric acid. When chloride is tested using these sample and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.02 % or less).
iv. Sulfate: 5.0 g (4.95~5.04 g) of this product is weighed to prepare a sample solution
using the sulfate test method. A control solution is prepared using 3.10 mL of 0.005
mol/L sulfuric acid. When sulfate is tested using these sample and control solutions,the turbidity of the sample solution shall not be higher than that of the control solution(0.03 % or less).
v. Lead: 5.0 g (4.95~5.04 g) of this product is weighed. When lead is tested using thelead test method (Method No. 1 of the atomic absorption spectrophotometry), theamount of lead shall be 1 μg/g or less. In so doing, 0.5 mL of lead standard solution ismeasured using a volumetric pipette and put in a 10 mL volumetric flask, added withnitric acid (1 → 150) up to the capacity mark to make 10 mL. This is used as astandard solution.
vi. Potassium: 5.0 g (4.95~5.04 g) of this product is weighed in a platinum crucible,
gently heated to carbonize, and heated at 500 °C or less to incinerate. The residue is314

Provisional Translation from Japanese Originaltransferred into a 100 mL tall form beaker with a small amount of water, 10 mL ofhydrochloric acid is gradually added, boiled for several minutes and allowed to cool.
Transfer this solution into a 250 mL volumetric flask with water, added with water up
to the capacity mark, and filtered with filter paper. 50 mL of the filtrate is put in a 100mL volumetric flask using a volumetric pipette. 10 mL of interference suppression
agent solution is added, and water is added up to the capacity mark to make 100 mL.
Using this as a sample solution, a test is performed by atomic absorption spectrometry(flame type).
Simultaneously, 10 mL of hydrochloric acid is put in a 250 mL volumetric flask,
and added with water up to the capacity mark to make 250 mL. 50 mL of the filtrate ismeasured using a volumetric pipette and put in a 100 mL volumetric flask. 10 mL ofinterference suppression agent solution is added, and added with water up to thecapacity mark to make 100 mL. A blank test is performed using this solution andcorrections are made.
Separately, 2 mL of potassium standard solution is measured using a volumetricpipette and put in a 100 mL volumetric flask. 10 mL of interference suppression agent
solution is added, and added with water up to the capacity mark to make 100 mL. Thisis used as a standard solution.
When the sample and standard solutions are tested by atomic absorption
spectrophotometry at the wavelength of 766.5 nm, using a potassium hollow cathodelamp as the light source, acetylene as a flammable gas, and air as a combustionsupporting gas, the absorbance of the sample solution shall be equal to or lower thanthat of the standard solution (0.2 % or less).
Preparation of interference suppression agent solution: 12.5 g (12.45~12.54 g) of
calcium carbonate [special grade] (CaCO3) is weighed and put in a beaker, added witha small amount of water, and added with 105 mL of hydrochloric acid gradually. Thissolution is boiled, allowed to cool, and added with water to make 1,000 mL.
vii. Sodium: The sample solution obtained in vi. is tested by atomic absorption
spectrometry (flame type). A blank test is performed in the same way as vi. andcorrections are made. Separately, 1 mL of sodium standard solution is measured using
a volumetric pipette and put in a 100 mL volumetric flask, and added with water up to
the capacity mark. This is used as a standard solution. When the sample and standard
solutions are tested by atomic absorption spectrophotometry at the wavelength of
589.0 nm, using a sodium hollow cathode lamp as the light source, acetylene as aflammable gas, and air as a combustion-supporting gas, the absorbance of the sample
solution shall be equal to or lower than that of the standard solution (0.1 % or less).

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Provisional Translation from Japanese Original

Water content: 4.0 % or less (direct titration)
Ignition residue: 0.1 % or less (1 g)Assay: Approximately 3.0 g of this product is weighed to the digit of 0.001 g and the value
is recorded. It is dissolved with 40 mL of acetone and 20 mL of water and titrated with 1
mol/L hydrochloric acid (potentiometric titration). A blank test is performed in the same
way and corrections are made.
1 mol/L hydrochloric acid 1 mL = 161.20 mg C7H15NO3(b) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is a powder mixture of the raw material for manufacturing of L-Carnitine
and hydrated amorphous silicon oxide.
Content: When this product is determined, it contains L-carnitine (C7H15NO3)
corresponding to 90~110 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of LCarnitine is applied mutatis mutandis.
Assay: The assay of the raw material for manufacturing of L-Carnitine is applied mutatis
mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-Carnitine isapplied mutatis mutandis.

(40) β-Carotene
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried in a desiccator (reduced pressure,silica gel) for 4 hours, it contains 98.0 % or over of β-carotene (C40H56).
Physical and chemical properties: This product is reddish violet to dark red crystals or
crystalline powder with a slight, specific odor and taste.
Confirmation test:
i. 10 mL of a solution of this product in chloroform (1 → 1,000) is orange. When thissolution is added with 1 mL of antimony trichloride test solution, it is blue-green.

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Provisional Translation from Japanese Original

ii. The sample solution obtained by the assay is measured for its absorption spectrum
using cyclohexane as a control. The absorption maximum is at wavelengths of454~457 nm and 481~484 nm.
Purity test:
i. Melting point: 176~183 °C (degradation)
ii. Clarity and color of solution: 0.1 g (0.05~0.14 g) of this product is dissolved with 10
mL of chloroform. The resulting solution shall be dark red and clear.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
v. Absorption ratio: For the sample stock solution and the sample solution obtained by
the assay, the absorptions A1 and A2 at the wavelengths of 340 and 362 nm of thesample stock solution and absorptions A3, A4 and A5 at the wavelengths of 434, 455
and 483 of the sample solution are measured using cyclohexane as a control. A2/A1,(A4 × 10)/A1, A4/A3, and A4/A5 shall be 1.00 or more, 15.0 or more, 1.30~1.60, and1.05~1.25, respectively.
Loss on drying: 1.0 % or less (1 g, silica gel, reduced pressure, 4 hours)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried in a desiccator (reduced pressure, silica gel) for 4 hours,
approximately 0.04 g of it is weighed to the digits of 0.1 mg, and the value is recorded. It
is dissolved with 10 mL of chloroform, transferred to a 100 mL volumetric flask, andadded with cyclohexane to the graduation line to make 100 mL. 5 mL of this solution isweighed using a volumetric pipette into a 100 mL volumetric flask, and added withcyclohexane to the graduation line to make 100 mL. This is used as a sample stock
solution. 10 mL of the sample stock solution is measured using a volumetric pipette into a
100 mL volumetric flask, and added with cyclohexane to the graduation line to make 100
mL. This is used as a sample solution. The absorbance A of this solution is measured at
the maximum wavelength at around wavelength 455 nm using cyclohexane as a control.
Amount of β-carotene (C40 H56) (mg) =A× 200,000
2,450

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Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in a lightproof capped container in which air is replaced with inert gas.
B. Preparation(a) Compositional standards
This product is particles of the mixture of the raw material for manufacturing of βcarotene and excipient.
Content: When this product is determined, it contains β-carotene (C40H56) corresponding to
90~130 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.01 g of
the raw material for manufacturing of β-carotene is weighed, added with 100 mg(99.5~100.4 mg) of trypsin, added with 3 mL of water, occasionally shaken up in awater bath at 40 °C for 5 minutes, sonicated for 10 minutes to be suspended if theparticles remain. It is allowed to cool, added with 20 mL of chloroform, shaken up
well, and centrifuged for 5 minutes at 3,000 rpm, and then the chloroform layer isorange. When 2 mL of the chloroform layer is measured, added with 0.5 mL ofantimony trichloride test solution, and allowed to stand, the solution is blue-green.
ii. The sample solution obtained by the assay is measured for its absorption spectrum
using cyclohexane as a control. The absorption maximum is at wavelengths of454~457 nm and 481~484 nm.
Assay: The amount of this product containing approximately 5 mg of β-carotene (C40H56) isweighed to three significant digits and the value is recorded. It is transferred to a 100 mL
volumetric flask, added with 100 mg (99.5~100.4 mg) of trypsin, added with 3 mL ofwater, occasionally shaken up in a water bath at 40 °C for approximately 5 minutes. If theparticles remain, it is sonicated for 10 minutes to be suspended. It is allowed to cool,
added with 30 mL of absolute ethanol, shaken up well, and added with ether to thegraduation line to make 100 mL. This solution is shaken up well, and some of it iscentrifuged for 5 minutes at 3,000 rpm. 2 mL of the supernatant is measured using a
volumetric pipette and the solvent is distilled away in a water bath at 50 °C under
nitrogen stream. The residue is dissolved with approximately 0.5 mL each of absoluteethanol and chloroform, transferred to a 50 mL volumetric flask, added with cyclohexane
to the graduation line to make 50 mL. This is used as a sample solution. The absorbance
A of the sample solution of the maximum wavelength at wavelength around 455 nm is
measured using cyclohexane as a control.
Amount of β-carotene (C40 H56) (mg) =A× 25,000
2,230

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Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in a lightproof airtight container.

(41) Canthaxanthin
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 96.0~102.0 % of canthaxanthin(C40H52O2).
Physical and chemical properties:
i. This product is reddish violet to dark reddish violet crystalline powder.
ii. This product is easy to dissolve in chloroform and hardly dissolves in absolute ethanol,
ether, cyclohexane and water.
iii. This product gradually changes by oxygen and light.
iv. Melting point: About 209 °C (degradation)
Confirmation test:
i. When 0.5 mL of a solution of this product in chloroform (1 → 10,000) is added with 1
mL of antimony trichloride test solution, the resulting solution is dark blue.
ii. The sample solution obtained by the assay is measured for its absorption spectrum
using cyclohexane as a control. The absorption maximum is at wavelengths of
470~476 nm.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, reduced pressure, silica gel, 3 hours)
Ignition residue: 0.1 % or less (1 g)Assay: Approximately 10 mg of this product is weighed to the digits of 0.1 mg and thevalue is recorded. It is dissolved with chloroform, transferred to a 200 mL volumetricflask, and added with chloroform to the graduation line to make 200 mL. 4 mL of this319

Provisional Translation from Japanese Original

solution is measured using a volumetric pipette to a 100 mL volumetric flask, and addedwith cyclohexane to the graduation line to make 100 mL. This is used as a sample
solution. The absorbance A of this solution at the maximum wavelength at aroundwavelength 473 nm is measured using cyclohexane as a control.
Amount of canthaxanthin (C40 H52 O2 ) (mg) =A× 50,000
2,200(b) Standard of storage method

It shall be stored in a lightproof airtight container in which air is replaced with nitrogen gas.
B. Preparation(a) Compositional standards
This product is particles of the mixture of the raw material for manufacturing of
canthaxanthin and excipient.
Content: When this product is determined, it contains canthaxanthin (C40H52O2)
corresponding to 90~120 % of the amount on the label.
Confirmation test: 5 mL of the filtrate obtained by the assay is measured and the solvent isdistilled away in a water bath at 50 °C under nitrogen stream. The residue is dissolved
with 5 mL of chloroform to prepare a sample solution. 10 µL of this solution is spotted ona thin layer plate prepared using silica gel for thin-layer chromatography. Then it isdeveloped approximately 15 cm with the developing solvent, a mixture ofdichloromethane and ether (9:1) in a dark place. When the thin layer plate is air dried in adark place, the canthaxanthin spot obtained from the sample solution is red and its Rfvalue is approximately 0.4.
Assay: The amount of this product containing approximately 10 mg of canthaxanthin(C40H52O2) is weighed to three significant digits and the value is recorded. It is addedwith 10 mL of water warmed at 60 °C, warmed in a water bath at 60 °C, and stirred under
nitrogen stream to make it a complete suspension. It is allowed to cool, transferred to a
250 mL volumetric flask and added with 100 mL of absolute ethanol. Then it is addedwith chloroform to the graduation line to make 250 mL and filtered. 20 mL of the first
filtrate is removed, 5 mL of the next filtrate is measured using a volumetric pipette to a
100 mL round-bottom flask, and the solvent is distilled away in a water bath at 50 °C
under nitrogen stream. The residue is moisturized with several drops of absolute ethanol,
added with 50 mL of cyclohexane, and allowed to stand at 50 °C for approximately 2
minutes to dissolve completely. This solution is allowed to cool, transferred to a 100 mL
volumetric flask, added with cyclohexane to the graduation line to make 100 mL. This isused as a sample solution. The absorbance A of this solution at the maximum wavelength
at around wavelength 470 nm is measured using cyclohexane as a control.

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Provisional Translation from Japanese OriginalAmount of canthaxanthin (C40 H52 O2 ) (mg) =A× 50,000
1,970(b) Standard of storage method

It shall be stored in a lightproof airtight container.

(42) Guanidinoacetic Acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined,it contains guanidinoacetic acid (C3H7N3O2)
at no less than 96.5 %.
Physical and chemical properties:
i. This product is white to light brown powder.
ii. The pH of an aqueous suspension of this product (1 → 10) is 8.0~9.0.
iii. This product is slightly insoluble in water, and extremely insoluble in ethanol.
Confirmation test:
i. When 1 mL of ninhydrin test solution is added to 5 mL of an aqueous solution of thisproduct (1 → 1,000) and heated for 3 minutes, the solution does not develop a color.
ii. When 1 mL of 8-oxyquinoline test solution and 1 mL of N-bromosuccinimide solution(1 → 1,000) are added to 2 mL of an aqueous solution of this product (1 → 20,000),
the solution develops a red-yellow color.
Purity test:i. Lead: Weigh 2.0 g (1.95~2.04 g) of this product and when a test for lead is performedby the lead limit test (atomic absorption spectrometry No.1), the amount shall be no
more than 5 μg/g.
ii. Arsenic: Weigh 1.0 g (0.95~1.04 g) of this product, prepare a sample solution byaccording to the arsenic limit test method3, and when a test for arsenic is performed by
the method using apparatus A, the color of absorption solution shall not be deeper than
the standard color (no more than 2 μg/g).
iii. Dicyandiamide: Weigh 0.500 g (0.4995~0.5004 g) of this product, add water todissolve with sonicating if necessary, place in a 1,000 mL volumetric flask, further add
water to the capacity mark (i.e. volume up to 1,000 mL), filter with a membrane filter

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Provisional Translation from Japanese Original(0.45 μm), and serve the filtrate as the sample solution. Separately, weigh 10.0 mg(9.95~10.04 mg) of dicyandiamide reference standard, add water to dissolve, place in a
1,000 mL measuring flask, and further add water, to the capacity mark (i.e. volume up
to 1,000 mL). Measure 25 mL of this solution using a volumetric pipette, place in a
100 mL measuring flask, and add water to the capacity mark (i.e. volume up to 100mL) to prepare the standard solution. Measure 20 μL each of the sample solution andstandard solution and when a test is performed by liquid chromatography under theconditions below, the peak area of dicyandiamide in the sample solution shall be no
more than the peak area of dicyandiamide in the standard solution multiplied by the
purity of dicyandiamide reference standard (no more than 0.5 %).
Operating condition
Detector: Ultraviolet absorptiometer (measuring wavelength: 220 nm)
Column: Fill a stainless steel tube of 4.6 mm in inner diameter and 250 mm inlength with octadecylsilyl silica gel for liquid chromatography of 5 μm in
particle diameter.
Mobile phase: Water
Flow rate: Approximately 1.5 mL per minute.
iv. Cyanamide: Weigh 0.400 g (0.3995~0.4004 g) of this product, add water to dissolvewith sonicating if necessary, place in a 100 mL volumetric flask, further add water tothe marked line (i.e. volume up to 100 mL), filter with a membrane filter (0.45 μm),
and serve the filtrate as the sample solution. Separately, weigh 24.0 mg (23.95~24.04mg) of cyanamide reference standard, add water to dissolve, place in a 1,000 mL
measuring flask, and further add water to the capacity mark (i.e. volume up to 1,000mL). Measure 10 mL of this solution using a volumetric pipette, place in a 200 mL
measuring flask, and add water to the marked line (i.e. volume up to 200 mL) to
prepare the standard solution. Measure 50 μL each of the sample solution and standard
solution and when a test is performed by liquid chromatography under the conditions
below, the peak area of cyanamide in the sample solution shall be no more than thepeak area of cyanamide in the standard solution multiplied by the purity of cyanamidereference standard (no more than 0.03%).

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Provisional Translation from Japanese Original

Operating condition
Detector: Ultraviolet spectrophotometer (measuring wavelength: 210 nm)
Column: Connect the columns i to iii below in this order, and use i at the sample
injection device side and iii at the detector side.
i:

Fill a resin tube of 4.6 mm in inner diameter and 100 mm in length withporous graphite carbon for liquid chromatography of 7 μm in particle
diameter.

ii: Fill a resin tube of 4 mm in inner diameter and 50 mm in length withanion exchange resin for liquid chromatography of 9 μm in particle
diameter.
iii: Fill a resin tube of 4 mm in inner diameter and 250 mm in length withanion exchange resin for liquid chromatography of 9 μm in particle
diameter.
Column temperature: A constant temperature around 30 °C
Mobile phase: Add 4 mL of 1-mol/L sodium hydroxide test solution to 1,000 mLof water.
Flow rate: 1.0 mL per minute
v. Melamine Weigh 0.100 g (0.0995~0.1004 g) of this product, place in a 50 mL
container for homogenizer, add 50 μL of internal standard solution using a micropipet,
further add 25 mL of water-acetonitrile mixture solution (1:1) and 10 mL of n-hexane,
agitate using a homogenizer for 30 seconds, centrifuge at 900×g for 5 minutes, andserve the lower layer as the extract.
Measure 1 mL of the extract using a micropipet, place in an ethylenediamine-npropylsilyl silica gel mini cartridge column previously washed serially with 5 mL of
methanol and 5 mL of water-acetonitrile mixture solution (1:1), then add 3 mL ofwater-acetonitrile mixture solution (1:1) and collect the eluate.
Add 130 μL of 1 mol/L hydrochloric acid test solution to this eluate, place it in astrongly acidic cation exchanger mini cartridge column previously washed seriallywith 5 mL of ammonia water-methanol mixture solution (1:19), 5 mL of methanol and
5 mL of water, add serially 2 mL of 0.1 mol/L hydrochloric acid test solution and 1 mL

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Provisional Translation from Japanese Originalof methanol, and discard the effluent. Elute using 5 mL of ammonia water-methanol
mixture solution (1:19), distill the solvent off in a nitrogen stream at 45°C, add 2 mLof formic acid (1 → 1,000)-acetonitrile mixture solution (1:1) to the residue, andsonicate for 5 seconds to prepare the sample solution.
Separately, weigh 0.01 g (0.0095~0.0104 g) of melamine reference standard, add
water-acetonitrile mixture solution (1:1) to dissolve, place in a 100 mL volumetricflask, and further add water-acetonitrile mixture solution (1:1) to the marked line (i.e.
volume up to 100 mL) to prepare the standard stock solution.
Accurately dilute certain quantities of the standard stock solution with formic acid
(1 → 1,000)-acetonitrile mixture solution (1:1) to prepare some solutions containing
amounts equivalent to 0.0025, 0.005, 0.01, 0.025 and 0.05 μg in 1 mL. Add an internal
standard solution at a rate of 1 μL per 1 mL of each solution to prepare the standard
solutions.
For 1 μL of the sample solution and standard solutions, when test for melamine is
performed by liquid chromatography / mass spectrometry under the conditions below,
the amount shall be no more than 20 μg/g.
Operating condition
Detector: Mass spectrometer (two mass spectrometry parts shall be seriallyconnected, and a collision cell shall be placed in between)
Column: Fill a stainless steel tube of 2 mm in inner diameter and 150 mm inlength with a column packing material in which carbamoyl group is
introduced to the surface of silica gel substrate of 3 μm in particle diameter.
Mobile phase: Add 1,000 mL of water to 0.77 g (0.765~0.774 g) of ammonium
acetate to dissolve, and serve it as Solution A. Solution B is acetonitrile, andfeed under the conditions below.
0~5 min

Solution B at 95%

5~10 minSolution B at 95% → Solution B at 90%

10~20 min Solution B at 90%
Flow rate: 0.2 mL per minute
Ionization method: Electrospray ionization method (cation detection mode)324

Provisional Translation from Japanese Original

Measurement mode: Selected reaction monitoring
Set mass number: m/z 127 → 85 (melamine quantification ion)m/z 127 → 68 (melamine confirmation ion)m/z 130 → 87 (internal standard melamine-15N3
quantification ion)
Preparation of internal standard solution Weigh 0.005 g (0.0045~0.0054mg) of melamine-15N3 reference standard, add water-acetonitrile mixture
solution (1:1) to dissolve, place in a 50-mL volumetric flask, and further
add water-acetonitrile mixture solution (1:1) to the marked line (i.e. volume
up to 50 mL) to prepare the internal standard stock solution. Accurately
dilute a certain quantity of the internal standard stock solution with wateracetonitrile mixture solution (1:1) to contain an amount equivalent to 10 μg
in 1 mL.
Ethylenediamine-n-propylsilyl silica gel mini cartridge column A
propropylene column tube filled with 500 mg of ethylenediamine-npropylsilyl silica gel or a column with equivalent separation characteristics.
Strongly acidic cation exchanger mini cartridge column A propropylene
column tube filled with 500 mg of strongly acidic cation exchanger or acolumn with equivalent separation characteristics.
Water content No more than 1.0 % (direct titration)
Quantification method Weigh approximately 10 mg of this product to the 0.01 mg digit,
record the value, add approximately 200 mL of water to dissolve with sonicating if
necessary, place in a 250-mL volumetric flask, further add water to the marked line (i.e.
volume up to 250 mL), filter with a membrane filter (0.45 μm) if necessary, and serve thefiltrate as the sample solution. For 10 μL of this solution, perform testing by liquid
chromatography under the conditions below. Calculate the peak area of guanidinoacetic
acid from the resulting chromatogram to calculate the concentration of guanidinoacetic
acid using a separately prepared calibration curve and calculate the content.
Operating condition
Detector: Ultraviolet spectrophotometer (measuring wavelength: 200 nm)

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Provisional Translation from Japanese Original

Column: Fill a stainless steel tube of 4.6 mm in inner diameter and 250 mm inlength with a column packing material for hydrophilic interaction
chromatography of 5 μm in particle diameter.
Column temperature: A constant temperature around 30°C
Mobile phase: Water-acetonitrile mixture solution (3:7)
Flow rate: Approximately 1.0 mL per minutePreparation of calibration curve
Weigh approximately 25 mg of guanidinoacetic acid reference standard to the0.01 mg digit, record the value, add approximately 200 mL of water to dissolvewith sonicating if necessary, place in a 250 mL volumetric flask, and further add
water to the marked line (i.e. volume up to 250 mL) to prepare the standard stock
solution. Add water to certain quantities of the standard stock solution ,accurately

dilute the solutions to contain amounts equivalent to 1, 3, 10 and 50 μg in 1 mL,
filter the solutions and standard stock solution with a membrane filter (0.45 μm) if
necessary, and serve the filtrate as standard solutions. For 10 μL each of thestandard solutions, perform testing by liquid chromatography in the same manneras the case for sample solution. Calculate the peak area of guanidinoacetic acidfrom the resulting chromatogram to prepare a calibration curve.
(b) Standard of storage method
Shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is powder or granule in which starch is mixed into source material for
manufacturing guanidinoacetic acid.
Content When quantified, this product contains guanidinoacetic acid (C3H7N3O2) at noless than 95.5 %.
Confirmation Apply mutatis mutandis the confirmation for source material for
manufacturing guanidinoacetic acid.
Quantification method Apply mutatis mutandis the quantification method for source
material for manufacturing guanidinoacetic acid.
(b) Standard of storage method326

Provisional Translation from Japanese Original

Apply mutatis mutandis the standards for methods of storage for source material for
manufacturing guanidinoacetic acid.

(43) Ferric Citrate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 16.5~18.5 % of iron (Fe).
Physical and chemical properties:
i. This product is red-brown, transparent small lamina or brown powder.
ii. This product is slightly hard to dissolve in cold water, easy to slightly dissolve in hot
water, and hardly dissolves in ethanol.
iii. The pH of a solution (1 → 20) of this product in water is 1.0~2.0.
iv. A solution of this product in water is gradually reduced by light to become ferrous
citrate.
Confirmation test:
i. 10 mL of a solution of this product in water (1 → 20) gives the qualitative reaction i. of
ferric salt.
ii. 10 mL of a solution of this product in water (1 → 20) is added with 15 mL of 1 mol/L
sodium hydroxide test solution and heated on a water bath for 10 minutes, with stirringwell. It is allowed to cool and is filtered. 10 mL of the filtrate is neutralized with acetic
acid. The resulting solution gives the qualitative reaction iii. of citrate.
Purity test:
i. Clarity and color of solution: 1 g (0.5~1.4 g) of this product is added with 20 mL ofwater and heated in a water bath to dissolve. The resulting solution shall be almost
clear or clear.
ii. Lead: When 0.5 g (0.45~0.54 g) of this product is weighed and subjected to the leadtest method (Method No. 1 of atomic absorption spectrophotometry), the content oflead shall be 20 mg/kg or less.
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Assay: Approximately 1.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is placed in a stoppered flask, added with 5 mL of hydrochloric acid and 30
mL of water, and dissolved by heating. It is allowed to cool, added with 4.0 g (3.95~4.04

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Provisional Translation from Japanese Originalg) of potassium iodide, tightly stoppered, allowed to stand for 15 minutes in a dark place,added with 100 mL of water, and titrated with 0.1 mol/L sodium thiosulfate solution(indicator: 1 mL of starch test solution). A blank test is performed in the same way andcorrections are made.
0.1 mol/L sodium thiosulfate solution 1 mL = 5.585 mg Fe(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of ferric citrate areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ferric citrate isapplied mutatis mutandis.

(44) Calcium gluconate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 80 °C for 2 hours, it contains
98.0~104.0 % of calcium gluconate (C6H12CaO14·H2O).
Physical and chemical properties: This product is white crystalline or particulate powder.
Confirmation test:
i. A solution of this product in water (1 → 40) gives the qualitative reaction of calcium
salt.
ii. When 1 mL of a solution of this product in water (1 → 40) is added with a drop of
ferric chloride test solution, the resulting solution is deep yellow.
iii. 5 mL of a solution of this product in water (1 → 10) is added with 0.7 mL of acetic
acid and 1 mL of newly distilled phenylhydrazine, heated on a water bath for 30
minutes and allowed to cool. The inner wall is rubbed with a glass bar, and thencrystals are deposited. The crystals are collected by filtration, dissolved with 10 mL ofboiling water, added with a little activated charcoal, and filtered. When it is allowed tocool, the inner wall is rubbed with a glass bar, the deposited crystals are dried, and themelting point is 192~202 °C (degradation).

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Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is added with 20
mL of water and dissolved by warming, the resulting solution shall be colorless andalmost clear.
ii. pH: The pH of a solution of this product in water (1 → 20) shall be 6.0~8.0.
iii. Chloride: 0.30 g (0.295~0.304 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.60 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.071 % or less).
iv. Sulfate: 0.50 g (0.495~0.504 g) of this product is weighed to prepare a sample
solution by the sulfate test method. A control solution is prepared using 0.50 mL of
0.005 mol/L sulfate. When sulfate is tested using these samples and control solutions,the turbidity of the sample solution shall not be higher than that of the control solution(0.048 % or less).
v. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
vi. Arsenic: 0.50 g (0.495~0.504 g) of this product is weighed, added with 5 mL of water,and dissolved by warming. The resulting solution is added with 5 mL of sulfuric acid
(3 → 50) and 1 mL of bromine test solution and heat-concentrated on a water bath to
make 5 mL. This is used as a sample solution. For the sample solution the arsenic test
is performed using the method using device A. The color of absorbing solution shallnot be darker than the standard color (4 mg/kg or less).
vii. Sucrose or reducing sugar: 0.50 g (0.495~0.504 g) of this product is weighed, addedwith 10 mL of water and 2 mL of hydrochloric acid (1 → 4) and boiled for 2 minutes.
It is allowed to cool, added with 5 mL of anhydrous sodium carbonate solution (1 →8), allowed to stand for 5 minutes, and added with water to make 20 mL. When 5 mLof this solution is measured, added with 2 mL of Fehling's test solution, boiled for aminute, orange-yellow to red precipitation shall not be generated within 30 seconds.
Loss on drying: 0.50 % or less (1 g, 80 °C, 2 hours)
Assay: This product is dried at 80 °C for 2 hours. Approximately 0.4 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved with 100 mL of water,
added with 10 mL of potassium hydroxide solution (1 → 10), allowed to stand for329

Provisional Translation from Japanese Original

approximately a minute, added with 0.1 g of NN indicator, and titrated with 0.05 mol/L
ethylene-diamine-tetraacetic acid disodium solution within 30 seconds. In this case, theend point of the titration is the point at which the red color of the solution completely
disappears and changes to blue.
0.05 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL
= 22.42 mg C6H12CaO14·H2O(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium gluconate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium
gluconate is applied mutatis mutandis.

(45) Monosodium L-glutamate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 100 °C for 5 hours, it contains
99.0 % or over of monosodium L-glutamate (C5H8NNaO4·H2O).
Physical and chemical properties:
i. This product is colorless to white pillar crystals or white crystalline powder with a
specific taste.
ii. The pH of a solution of this product in water (1 → 10) is 6.7~7.2.
Confirmation test:
i. 5 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes. The resulting solution is purple.
ii. A solution of this product in water (1 → 10) gives the qualitative reaction of sodium
salt.
Purity test:i. Specific rotation: Approximately 5 g of this product is weighed to the digits of 0.01 g,
and the value is recorded. It is dissolved in 2.5 mol/L hydrochloric acid test solution to
make 50 mL. The rotation of this solution measured at a path length 100 mm shall be[α]20
D = +24.8 ~ +25.3°

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Provisional Translation from Japanese Originalii. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved in 10 mLof water. The resulting solution shall be colorless and clear.
iii. Chloride: 0.10 g (0.095~0.104 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.2 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.071 % or less).
iv. Ammonium salt: The purity test iii. for the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis (0.02 % or less).
v. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 1.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
vi. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, 100 °C, 5 hours)
Assay: This product is dried at 100 °C for 5 hours, approximately 0.15 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (indicator: 0.5 mL of α-naphtholbenzein test solution). In this case,the end point of the titration is the point at which the color of the solution changes frombrown to green. A blank test is performed in the same way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 9.357 mg C5H8NNaO4·H2O(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of monosodium Lglutamate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of monosodium Lglutamate is applied mutatis mutandis.

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Provisional Translation from Japanese Original(46) Iron and Sodium Succinate Citrate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 10.0~11.0 % of iron (Fe).
Physical and chemical properties:
i. This product is blue-white to green-whitish powder with no odor.
ii. This product is easy to slightly dissolve in water and hardly dissolves in ethanol.
iii. This product is stable against heat but gradually oxidized by light and becomesbrown.
Confirmation test:
i. A solution of this product in water (1 → 100) gives the qualitative reaction of ferrous
salt.
ii. When 5 mL of a solution of this product in water (1 → 100) is added with 2 mL ofstrong ammonia solution, the resulting solution is red-brown and precipitation is not
generated.
iii. The residue obtained by the ashing of 3.0 g (2.95~3.04 g) of this product gives thequalitative reaction of sodium salt.
iv. 0.5 g (0.45~0.54 g) of this product is added with 5 mL of water and 10 mL ofpotassium hydroxide test solution and heated on a water bath for 10 minutes with
sufficient stirring. It is allowed to cool and is filtered. 10 mL of the filtrate is measured
and neutralized with acetic acid. The resulting solution gives the qualitative reactioniii. of citrate.
v. 0.2 mL of the solution neutralized with acetic acid in iv. is measured into a porcelainpot, added with a drop of dilute hydrochloric acid to make it acidic, and evaporated todryness. The residue is added with 5 drops of thionyl chloride and heated to almost
dryness. Then, it is added with 5 drops of saturated ethanol solution of hydroxylaminehydrochloride and is further added with dilute potassium hydroxide-ethanol test
solution to make it alkaline. It is boiled for approximately 30 seconds, added with adrop of dilute hydrochloric acid to make it acidic, and added with a drop of ferricchloride test solution. The resulting solution is dark purple.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is added with 10 mLof water and dissolved by warming. The resulting solution shall be dark green-brown
and clear.
ii. Lead: 2.0 g (1.95~2.04 g) of this product is weighed, added with 4 mL of nitric acidand 30 mL of water, boiled for 10 minutes, allowed to cool, transferred to a 50 mL

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Provisional Translation from Japanese Original

volumetric flask, and added with water to the graduation line to make 50 mL. 25 mLof this solution is measured using a volumetric pipette, and this is used as a sample
solution. When lead is tested using the lead test method (dithizone method) for the
sample solution, the amount shall be 10 mg/kg or less.
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
iv. Ferric salt: 2.0 g (1.95~2.04 g) of this product is placed in a stoppered flask, dissolved
with 5 mL of hydrochloric acid and 30 mL of water, added with 4.0 g (3.95~4.04 g) ofpotassium iodide. The flask is closed with a stopper and placed for 15 minutes in adark place. When the solution is added with 2 mL of starch test solution and shaken up
well, it gives color, but the color shall disappear when it is added with 1 mL of 0.1
mol/L sodium thiosulfate.
Assay: Approximately 1.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is placed in an iodine bottle, added with 25 mL of dilute sulfuric acid and 2
mL of nitric acid, boiled for 10 minutes, allowed to cool, added with 20 mL of water and
4.0 g (3.95~4.04 g) of potassium iodide. The iodine bottle is closed with a stopper andplaced in a dark place for 15 minutes. Then the solution is added with 100 mL of waterand titrated with 0.1 mol/L sodium thiosulfate solution (indicator: 3 mL of starch test
solution). A blank test is performed in the same way and corrections are made.
0.1 mol/L sodium thiosulfate solution 1 mL = 5.585 mg Fe(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of iron and sodium
succinate citrate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of iron and sodium
succinate citrate is applied mutatis mutandis.

(45) Cholecalciferol
A. Raw material for manufacturing(a) Compositional standards

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Physical and chemical properties:
i. This product is white crystals with no odor.
ii. This product is easy to dissolve in ethanol or chloroform and easy to slightly dissolvein fatty oil and hardly dissolves in water.
iii. This product changes by air or light.
Confirmation test: The confirmation test for the raw material for manufacturing ofergocalciferol is applied mutatis mutandis. In this case, “147~149 °C” shall be replacedby “133~135 °C”.
Purity test:
i. Absorbance: The purity test i. of the raw material for manufacturing of ergocalciferol isapplied mutatis mutandis. In this case, “445~485” shall be replaced by “450~490”.
ii. Specific rotation: The purity test ii. of the raw material for manufacturing ofergocalciferol is applied mutatis mutandis. In this case, “0.3 g” and “+102 ~ +107°”
shall be replaced by “0.1 g” and “+103 ~ +112°”.
iii. Melting point: The purity test iii. of the raw material for manufacturing ofergocalciferol is applied mutatis mutandis. In this case, “115~118 °C” shall be
replaced by “84~88 °C”.
iv. 7-dehydrocholesterol: When 0.01 g (0.0095~0.0104 g) of this product is dissolved
with 2.0 mL of absolute ethanol (9 → 10), added with a solution of 0.02 g(0.0195~0.0204 g) of digitonin in 2.0 mL of absolute ethanol (9 → 10), and is allowed
to stand for 18 hours, precipitation shall not be generated.
(b) Standard of storage method
It shall be stored in a lightproof capped container in which air is replaced with nitrogengas. This product shall be stored at a cool place.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of cholecalciferol and hydrogenated oils, higher saturated fatty acids, fatty
acids, vegetable oil or animal fats.
Content: When this product is determined, it contains cholecalciferol (C27H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder are applied mutatis mutandis.
Assay: The test is performed by vitamin D determination method.

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Provisional Translation from Japanese Original(b) Standard of storage method
This product shall be stored in a lightproof sealed container in which air is replaced withnitrogen and shall be stored at a cool place.
C. Preparation (Part 2 powder)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof cholecalciferol and excipient.
Content: When this product is determined, it contains cholecalciferol (C27H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder are applied mutatis mutandis.
Assay: The test is performed by vitamin D determination method.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

(46) dl-α-Tocopherol Acetate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 96.0 % or over of dl-α-tocopherol
acetate (C31H52O3).
Physical and chemical properties:
i. This is colorless to yellow, clear, viscous fluid with no odor.
ii. This product is miscible with acetone, ether, chloroform, or vegetable oil.
iii. This product is easy to dissolve in ethanol and hardly dissolves in water.
iv. This product is not rotatory.
v. This product changes by air and light.
Confirmation test: When 10 mL of the sample solution obtained by the assay is added with
2 mL of nitric acid and heated at 75 °C for 15 minutes, the resulting solution is red toorange.
Purity test:
i. Clarity and color of solution: 0.1 g (0.05~0.14 g) of this product is dissolved withabsolute ethanol to make 10 mL. The resulting solution is clear and its color shall not
be darker than that of a solution prepared by which 0.5 mL of the color control stock
solution of ferric chloride is added with 0.5 mol/L hydrochloric acid test solution to
make 100 mL.

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Provisional Translation from Japanese Original

ii. Absorbance: 0.01 g of this product is weighed to the digits of 0.1 mg and the value isrecorded. It is dissolved in absolute ethanol to make 100 mL. The absorbance of thissolution measured at a wavelength of 284 nm shall be E1%
1 cm = 41.0~45.0.
iii. Refractive index: The refractive index, n20
D , of this product shall be 1.494~1.499.
iv. Specific gravity: The specific gravity, d20
20 , of this product shall be 0.952~0.966.
v. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
vi. Free-α-tocopherol: Approximately 1 g of this product is weighed to the digits of 0.01
g and the value is recorded. It is dissolved with 100 mL of a solution of sulfuric acid in
ethanol (3 → 200), added with 20 mL of water and titrated with 0.01 mol/L ceric
ammonium sulfate with sufficient stirring (indicator: 2 drops of diphenylamine test
solution). A blank test is performed in the same way and corrections are made. The
amount of free-α-tocopherol shall be 0.5 % or less. In this case, it must be noted thatthe assay is applied mutatis mutandis for the procedure.
0.01 mol/L ceric ammonium sulfate solution 1 mL = 2.154 mg C9H50O2
Assay: Approximately 0.25 g of this product is weighed to the digits of 0.001 g and thevalue is recorded. It is placed in a 100 mL brown round-bottom flask, dissolved with 25
mL of absolute ethanol, added with 20 mL of a solution of sulfuric acid in ethanol (3 →20), boiled for 3 hours with a reflux condenser. It is allowed to cool, transferred to a 200
mL brown volumetric flask, added with absolute ethanol to the graduation line to make200 mL. This is used as a sample solution. 50 mL of the sample solution is measured
using a volumetric pipette, added with 50 mL of a solution of sulfuric acid in ethanol (3→ 20) and 20 mL of water, and titrated with 0.01 mol/L ceric ammonium sulfate with
sufficient stirring (indicator: 2 drops of diphenylamine test solution). In this case, theprocedure shall be performed away from direct sunlight in a place as dark as possible.
The dropping rate shall be 25 drops per 10 seconds and the end point of the titration is thepoint at which the blue-violet color of the solution is maintained for 10 seconds. A blanktest is performed in the same way and corrections are made.
0.01 mol/L ceric ammonium sulfate solution 1 mL = 2.364 mg C31H52O3(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards

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Provisional Translation from Japanese Original

The compositional standards of the raw material for manufacturing of dl-α-tocopherol
acetate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of dl-α-tocopherol
acetate is applied mutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of dl-α-tocopherol acetate and hydrogenated oils, higher saturated fatty acids,
fatty acids, vegetable oil or animal fats.
Content: When this product is determined, it contains dl-α-tocopherol acetate (C31H52O3)
corresponding to 90~120 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.
Assay: The determination method of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of dl-α-tocopherol
acetate is applied mutatis mutandis.
D. Preparation (Part 3: powder)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof dl-α-tocopherol acetate and excipient.
Content: When this product is determined, it contains dl-α-tocopherol acetate (C31H52O3)
corresponding to 90~120 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.
Assay: The determination method of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

(49) Magnesium oxide
A. Raw material for manufacturing(a) Compositional standards

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Provisional Translation from Japanese OriginalContent: When this product is ignited at 1,000 °C for 30 minutes and then determined, it
contains 96.0 % or more of magnesium oxide (MgO).
Physical and chemical properties:
i. This product is white or whitish powder with no odor.
ii. This product is slightly dissolved in water resulting in weak alkaline.
iii. This product is easy to dissolve in dilute hydrochloric acid.
Confirmation test: A solution of 1 g (0.5~1.4 g) of this product in 25 mL of hydrochloric
acid (1 → 4) gives the qualitative reaction of magnesium salt.
Purity test:
i. Water-soluble matter: 2.0 g (1.95~2.04 g) of this product is weighed, added with 100
mL of water, heated in a water bath for 5 minutes, and filtered within 30 seconds.
After cooling, 25 mL of the filtrate is measured, evaporated to dryness in a water bath,and dried at 105 °C for an hour, and then the amount of the residue is 10 mg or less
(2 % or less).
ii. Hydrochloric acid-insoluble matter: 2.0 g (1.95~2.04 g) of this product is weighed,added with 75 mL of water, added by dropping hydrochloric acid until it no longer
dissolves while shaking it up, and is boiled for 5 minutes. It is allowed to cool and isfiltered. When the residue on the filter paper is washed with water until the washings
does not give the reaction of chloride and ignited together with the filter paper at
1,000 °C for 30 minutes, the amount of the residue is 20 mg or less (1 % or less).
iii. Free alkali: When 50 mL of the filtrate in i. is measured, added with 2 drops of methyl
red test solution, and is further added with 2.0 mL of 0.05 mol/L sulfuric acid, the redcolor of the solution does not disappear.
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with 25 mL ofhydrochloric acid (1 → 4), and concentrated in a water bath. At almost the end of
evaporation, the residue is stirred well to make it fine powder. It is dissolved with 20
mL of water, evaporated to dryness in the same way, and dissolved with 20 mL ofwater. As appropriate it is filtered, and added with 2 mL of acetic acid (1 → 20) andwater to make 50 mL. This is used as a sample solution. A control solution is preparedwith 2.0 mL of lead standard solution by Method No. 2 of the heavy metals test
method and the test for heavy metal is performed. The color of the sample solution
shall not be darker than that of the control solution (20 mg/kg or less).
v. Calcium oxide: 50 mL of solution A of the assay is measured, added with water tomake 300 mL, added with 10 mL of potassium hydroxide solution (1 → 10), allowed
to stand for a minute, and titrated with 0.01 mol/L ethylenediaminetetraacetic aciddisodium solution (indicator: approximately 0.1 g of NN indicator) using a338

Provisional Translation from Japanese Original

microburette, and its consumed volume shall be b mL. The end shall be the time when
the color of the sample solution, reddish violet, is completely consumed and changed
to blue. The content calculated using the following equation is 1.5 % or less.
Amount of calcium oxide (CaO) (%) =b (mL) × 0.5608
Amount of the sample weighed (g)vi. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and dissolved with 10 mL ofhydrochloric acid (1 → 4) to prepare a sample solution. For the sample solution, when
the arsenic test is performed by the method using device A, the color of absorbing
solution shall not be darker than the standard color (4 mg/kg or less).
Loss on ignition: 10.0 % or less (1 g, 1,000 °C, 30 minutes)
Assay: This product is dried at 1,000 °C for 30 minutes, approximately 0.5 g it is weighed tothe digits of 0.001 g, and the value is recorded. It is moisturized with 5 mL of water,
added with 10 mL of hydrochloric acid and 10 mL of perchloric acid, covered with awatch glass, gradually heated. After concentrated white smoke starts to be generated, it isheated for another 10 minutes. It is allowed to cool, added with approximately 50 mL ofwarm water and 5 mL of hydrochloric acid (1 → 2), heated a little, and filtered within 30
seconds. The filtrate is transferred to a 500 mL volumetric flask and added with water tomake 500 mL. This is used as solution A. 10 ml of solution A is measured using a
volumetric pipette, added with water to make 100 mL, added with 5 mL of pH 10.7
ammonia-ammonium chloride buffer and 2 drops of eriochrome black T test solution, andtitrated with 0.01 mol/L ethylenediaminetetraacetic acid disodium solution within 30
seconds, and the consumed volume, a mL, is determined. The end shall be the time when
the color of the sample solution changes from red to blue. The content is calculated using
the consumed volume, obtained in the purity test v., b mL, by the following equation.
Amount of magnesium oxide (MgO) (%) =(a - 0.2b) × 2.0152
Amount of the sample weighed (g)(b) Standard of storage method
It shall be stored in an airtight container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of magnesium oxide
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of magnesiumoxide is applied mutatis mutandis.

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Provisional Translation from Japanese Original(50) Cyanocobalamin
A. Raw material for manufacturing(a) Compositional standards
Content: By the determination, this product contains 95.0 % or more of cyanocobalamin(C63H88CoN14O14P) on the dried basis.
Physical and chemical properties:
i. This product is dark red crystals or powder.
ii. This product is hard to lightly dissolve in water, hard to dissolve in ethanol, and hardly
dissolves in acetone, ether, and chloroform.
iii. This product is hygroscopic.
Confirmation test:
i. In the measurement of the absorption spectrum of sample solution of this productprepared for the assay, the absorption maximum was shown at the wavelengths of277~279 nm, 360~362 nm, and 548~552 nm. When the absorbance at absorption
maximum each is defined as A1, A2, and A3, A1/A2 and A3/A2 are 0.53~0.59 and0.29~0.32, respectively.
ii. 1 mg (0.5~1.4 mg) of this product is mixed with 0.05 g (0.045~0.054 g) of potassiumacid sulfate, then ignited and melted. After cooling, the melt is crushed with a glassbar, added with 3 mL of water, dissolved by boiling, added with a drop ofphenolphthalein test solution, and added with 1 mol/L sodium hydroxide test solution
by dropping until the solution becomes pale red. Then, it is added with 0.5 g(0.45~0.54 g) of sodium acetate, 0.5 mL of diluted acetic acid, and 0.5 mL of 1nitroso-2-naphthol-3, 6-disulfonic acid disodium solution (1 → 500). The resulting
solution becomes red to orange-red within 30 seconds. The red color of the solution
does not disappear even when it is added with 0.5 mL more of hydrochloric acid andboiled for a minute.
iii. 5 mg (4.5~5.4 mg) of this product is placed in a 50 mL distilling flask, dissolved with
5 mL of water, and added with 2.5 mL of hypophosphorous acid. The flask is put witha short condenser and the tip of the condenser immersed in 1 mL of a sodiumhydroxide solution (1 → 50) in a test tube. Then it is gently boiled for 10 minutes anddistilled until the volume of distillate liquid reaches 1 mL. The solution in the test tube
is added with 4 drops of saturated solution of ammonium ferrous sulfate, gently shaken
up, added with 30 mg (25~34 mg) of sodium fluoride, heated until boiling, within 30
seconds added with sulfuric acid (1 → 7) by dropping until the solution becomes clear,
and added with 3~5 drops more of sulfuric acid (1 → 7). The resulting solution is blue
to blue-green.

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Provisional Translation from Japanese Original

Purity test: Pseudocyanocobalamin: 1.0 mg (0.95~1.04 mg) of this product is dissolved with
20 mL of water, transferred to a separatory funnel, added with 5 mL of a mixture of mcresol and carbon tetrachloride (1:1), vigorously shaken up for a minute, and allowed tostand. The bottom layer is transferred to another separatory funnel, added with 5 ml ofsulfuric acid (1 → 7) and then is vigorously shaken up. As appropriate, it is centrifuged,
and then the color of the supernatant shall be colorless, or shall not be darker than that ofa solution prepared by which 0.6 mL of 0.02 mol/L potassium permanganate solution isadded with water to make 1 L.
Loss on drying: 12 % or less (0.05 g, reduced pressure, 0.67 kPa or less, phosphorus
pentaoxide, 100 °C, 4 hours)
Assay: Approximately 0.02 g each of this product and the reference standard of
cyanocobalamin (the loss on drying is previously measured in the same way for thisproduct) is weighed to the digits of 0.1 mg, and the values are recorded. Each of them is
dissolved with water, transferred to a 1 L volumetric flask, added with water to thegraduation line to make 1 L. They are used as sample and standard solutions. The
absorbance, AT and AS at a wavelength of 361 nm of sample and standard solutions,
respectively, are measured.
Amount of cyanocobalamin (C63H88CoN14O14P) (mg) =Amount of the reference standard of cyanocobalamin on the dried basis (mg) ×ATAS(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof cyanocobalamin and excipient.
Content: When this product is determined, it contains cyanocobalamin (C63H88CoN14O14P)
corresponding to 90~120 % of the amount on the label.
Confirmation test: This product is made into powder. The residue obtained by pretreatment
by Method No. 1 of the assay for cyanocobalamin is placed in a centrifugal precipitation
tube for that which is granulated and coated with hydrogenated oils, higher saturated fatty
acids, fatty acids, vegetable oil or animal fats. For others, the amount containing 2 mg of
the raw material for manufacturing of cyanocobalamin is placed in a centrifugal
precipitation tube. It is added with 5 mL of water, vigorously shaken up for 10 minutes,
and centrifuged for 5 minutes. The supernatant is used as a sample solution. Separately, 2
mg (1.5~2.4 mg) of the reference standard of cyanocobalamin is weighed, dissolved with341

Provisional Translation from Japanese Original

5 mL of water to prepare a standard solution. 5 µL each of the sample and standard
solutions is spotted on a thin layer plate prepared using silica gel for thin-layer
chromatography. Then it is developed approximately 12 cm with the developing solvent,a mixture of methanol and water (19:1). When the thin layer plate is air dried, the spots
obtained from the sample and standard solutions are red and their Rf values are equal.
Assay: This product is made into powder and used as a sample. Method No. 2 is used for thecontent of cyanocobalamin on the label is 0.1 % or less, and Method No. 1 for others.
Provided, however that in the measurement of the absorption spectrum at wavelengths of
300~600 nm of the sample solution obtained by Method No. 1 using water as a control,when the absorption maximum is not at wavelengths of 360~362 nm or 548~552 nm, or
when the ratio of absorbances A1 and A2, A1/A2, is not within 0.29~0.32, Method No. 2 isused.
Method No. 1: For that which is granulated and coated with hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats, the amount of thisproduct containing approximately 2 mg of cyanocobalamin (C63H88CoN14O14P) is
weighed to three significant digits and the value is recorded. It is added with 50 mL ofchloroform, shaken up, and filtered using a glass filter (G4). The residue on the filter iswashed and filtered three times with 20 mL of chloroform, and air dried until thechloroform odor on the residue disappears. The residue is extracted and filtered while
adding 80 mL of water. The filtrate is placed in a 100 mL volumetric flask and addedwith water to the graduation line to make 100 mL. This is used as a sample solution.
For others, the amount of this product containing approximately 2 mg of
cyanocobalamin (C63H88CoN14O14P) is weighed to three significant digits and thevalue is recorded. It is placed in a 100 mL volumetric flask, added with 80 mL ofwater, shaken up well, added with more water to the graduation line to make 100 mL.
It is centrifuged for approximately 10 minutes at 3,000 rpm as appropriate, andfiltered. The first filtrate 20 mL is removed and the next filtrate is used as a sample
solution.
Separately, approximately 0.02 g of the reference standard of cyanocobalamin (theloss on drying is previously measured in the same way for the raw material for
manufacturing of cyanocobalamin) is weighed to the digits of 0.1 mg and the value isrecorded. It is dissolved with water, transferred to a 1 L volumetric flask, and addedwith more water to the graduation line to make 1 L. This is used as a standard
solution.
For each of them the assay of the raw material for manufacturing of cyanocobalamin ishereinafter applied mutatis mutandis.

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Provisional Translation from Japanese OriginalAmount of cyanocobalamin (C63H88CoN14O14P) (mg)
= Amount of the reference standard of cyanocobalamin on the dried basis (mg)
×

AT
1
×
AS 10

Method No. 2: The amount of this product containing approximately 2.5 mg of
cyanocobalamin (C63H88CoN14O14P) is weighed to three significant digits and thevalue is recorded. It is added with 10 mL of water using a volumetric pipette,
vigorously shaken up for 10 minutes, and centrifuged for 10 minutes at 2,700 rpm. 3
mL of the supernatant is measured using a volumetric pipette, added with 250 mg(249.5~250.4 mg) of DEAE-Sephadex A-25, allowed to stand for 30 minutes withoccasional shaking-up, and filtered using a membrane filter (0.8 µm). 400 µL of thefiltrate is measured using a micropipette, and spotted in a band shape at the height ofapproximately 2 cm from the lower end of a layer plate prepared using silica gel forthin-layer chromatography. Simultaneously, 5 µL of the standard solution of
cyanocobalamin (1 → 2,500) is spotted on the left side of the thin layer not to beoverlapped with the spot of the sample. Then, it is developed approximately 12 cm
with the developing solvent, a mixture of methanol and water (19:1) and the thin layer
plate is air dried. It is confirmed that the locations of the red spots of cyanocobalamin
obtained from the standard solution and the sample are consistent with each other, andthen the area at 1 cm up and down from the center of the band like spot obtained fromthe sample is scraped and placed in a glass column (inner diameter: 1 cm, length: 20cm) previously added with 5 mL of a mixture of methanol and water (19:1). The redcolor of cyanocobalamin is completely eluted using 50 mL of a mixture of methanol
and water (19:1). The eluate is collected, distilled using an aspirator in a water bath at
60 °C under reduced pressure with shaking. The residue is dissolved with water,transferred to a 5 mL volumetric flask, added with water to the graduation line to make
5 mL and filtered using a membrane filter (0.8 µm). The filtrate is used as a sample
solution. 1 g (0.5~1.4 g) of silica gel for thin-layer chromatography is weighed, placedin a glass column previously added with 5 mL of a mixture of methanol and water(19:1) and washed with 50 mL of a mixture of methanol and water (19:1). The
washings are collected, diluted using an aspirator in a water bath at 60 °C under
reduced pressure with shaking, added with water to make 5 mL, and filtered using amembrane filter (0.8 µm). The filtrate is used as a blank test solution. Separately,
approximately 0.02 g of the reference standard of cyanocobalamin (the loss on drying
is previously measured in the same way for the raw material for manufacturing of
cyanocobalamin) is weighed to the digits of 0.1 mg and the value is recorded. It is343

Provisional Translation from Japanese Original

dissolved with water, transferred to a 1 L volumetric flask, and added with more water
to the graduation line to make 1 L. This is used as a standard solution. The
absorbances, AT, AB, and AS, at the maximum wavelength at around wavelength 361
nm for the sample solution, the blank test solution, and the standard solution aremeasured, using water as a control.
Amount of cyanocobalamin (C63H88CoN14O14P) (mg)
= Amount of the reference standard of cyanocobalamin on the dried basis (mg)×AT - AB 1×AS8(b) Standard of storage method
It shall be stored in a lightproof capped container.

(51) Thiamine Mononitrate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 2 hours, it contains
98.0~102.0 % of thiamine mononitrate (C12H17N5O4S)
Physical and chemical properties:
i. This product is white to slightly yellow-white crystals or crystalline powder with noodor or a slight, specific odor.
ii. This product is slightly hard to dissolve in water, extremely hard to dissolve inethanol, and hardly dissolves in chloroform.
iii. Melting point: About 193 °C (degradation)
Confirmation test:
i. The confirmation tests i., ii. and iii. for the raw material for manufacturing of thiaminehydrochloride are applied mutatis mutandis.
ii. A solution of this product in water (1 → 50) gives the qualitative reactions i. and ii. of
nitrate.
Purity test:
i. pH: 1.0 g (0.95~1.04 g) of this product is dissolved with water to make 100 mL. The
pH of this solution shall be 6.5~8.0.
ii. Chloride: 0.20 g (0.195~0.204 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.3 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these sample and control

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solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.053 % or less).
iii. Sulfate: 1.5 g (1.45~1.54 g) of this product is dissolved with 30 mL of water and 2
mL of dilute hydrochloric acid and added with water to make 50 mL. This is used as asample solution. 0.35 mL of 0.005 mol/L sulfuric acid is added with 2 mL of dilutehydrochloric acid and water to make 50 mL. This is used as a control solution. Whensulfate is tested using these solutions, the turbidity of the sample solution shall not behigher than that of the control solution (0.011 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less). Provided, however, that the sample shall bewarmed to dissolve.
Loss on drying: 1.0 % or less (0.5 g, 105 °C, 2 hours)
Ignition residue: 0.20 % or less (1 g)Assay: This product and the reference standard of thiamine hydrochloride is dried at 105 °C
for 2 hours each. The assay for the raw material for manufacturing of thiaminehydrochloride is hereinafter applied mutatis mutandis.
Amount of thiamine nitrate (C12H17N5O4S) (mg)=Amount of the reference standard of thiamine hydrochloride (mg)×AT - AT'× 0.9706
AS - AS'(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of thiaminemononitrate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of thiaminemononitrate is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof thiamine mononitrate and excipient.

345

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains thiamine mononitrate (C12H17N5O4S)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.02 g of
the raw material for manufacturing of thiamine mononitrate is added with 50 mL ofwater and 10 mL of diluted acetic acid, and for a preparation containing hydrogenatedoils, higher saturated fatty acids, fatty acids, vegetable oil or animal fat, added with 20
mL of chloroform, shaken up, and allowed to stand, and the aqueous layer is filtered.
When 10 mL of the first filtrate is removed, and 5 mL of the next filtrate is measured
and added with 2~3 drops of Mayer's test solution, yellowish white precipitation or
opacity is generated.
ii. 1 mL of the filtrate of i. is measured and added with water to make 20 mL. 5 mL ofthis solution is measured and the confirmation test iii. for the raw material for
manufacturing of thiamine mononitrate is hereinafter applied mutatis mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forothers.
Method No. 1: The amount of this product containing approximately 0.02 g of thiaminemononitrate (C12H17N5O4S) is weighed to three significant digits and the value isrecorded. It is added with 50 mL of chloroform, shaken up, and filtered using a glass
filter (G4). The residue on the filter is washed three times with 20 mL each ofchloroform. When the chloroform odor on the residue disappears, the residue isfiltered while extracting with approximately 190 mL of warmed 0.1 mol/Lhydrochloric acid test solution, and allowed to cool. The filtrate is transferred to a 200mL volumetric flask and added with 0.1 mol/L hydrochloric acid test solution to thegraduation line to make 200 mL. 2 mL of this solution is measured using a volumetricpipette to a 100 mL volumetric flask and added with 0.001 mol/L hydrochloric acidtest solution to the graduation line to make 100 mL. This is used as a sample solution.
Separately, the reference standard of thiamine hydrochloride is dried at 105 °C for 2
hours, approximately 0.1 g of it is weighed to the digits of 0.001 g, and the value isrecorded. It is dissolved with 0.1 mol/L hydrochloric acid test solution, transferred to a
1 L volumetric flask, and is added with 0.1 mol/L hydrochloric acid test solution to thegraduation line to make 1 L. 2 mL of this solution is measured using a volumetricpipette to a 100 mL volumetric flask and is added with 0.001 mol/L hydrochloric acidtest solution to the graduation line to make 100 mL. This is used as a standard solution.
2 mL each of the sample solutions is measured using a volumetric pipette into

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stoppered centrifugal precipitation tubes T and T' and added with 3 mL each of acidic
potassium chloride test solution. The stoppered centrifugal precipitation tube T isadded with 3 mL of cyanogen bromide test solution, shaken up, added with 2 mL of
sodium hydroxide solution (3 → 10) within 30 seconds, shaken up, added with 15 mL
of isobutanol using a volumetric pipette, tightly stoppered, and vigorously shaken up
for 2 minutes. The stoppered centrifugal precipitation tube T' is added with 2 mL of
sodium hydroxide solution (3 → 10), shaken up, added with 3 mL of cyanogenbromide test solution, shaken up, added with 15 mL of isobutanol using a volumetricpipette, tightly stoppered, and vigorously shaken up for 2 minutes. Separately, 2 mL of
the standard solution is measured using a volumetric pipette into stoppered centrifugal
precipitation tubes S and S' and is subjected to the same procedure of the sample
solution. Each centrifugal precipitation tube is centrifuged for 2 minutes at a slow
speed. Then each isobutanol layer is collected into another tubes, as appropriate
gradually added with 1~2 g of anhydrous sodium sulfate, gently shaken up, andallowed to stand. The clear isobutanol solution is collected. As for each isobutanol
solution, the fluorescence intensities FT, FT', FS, and FS' are measured at an excitation
wavelength of approximately 370 nm and a fluorescence wavelength of approximately
440 nm.
Amount of thiamine mononitrate (C12H17N5O4S) (mg)
= Amount of the reference standard of thiamine hydrochloride (mg)×

FT - FT'× 0.1941
FS - FS'

Method No. 2: The amount of this product containing approximately 0.02 g of thiaminemononitrate (C12H17N5O4S) is weighed to three significant digits and the value isrecorded. It is added with 150 mL of warmed 0.1 mol/L hydrochloric acid test
solution, shaken up, and filtered. The residue on the filter is washed twice with 20 mL
each of 0.1 mol/L hydrochloric acid test solution. The filtrate and the washings arecollected together into a 200 mL volumetric flask and added with 0.1 mol/Lhydrochloric acid test solution to the graduation line to make 200 mL. 2 mL of thissolution is measured using a volumetric pipette into a 100 mL volumetric flask, addedwith 0.001 mol/L hydrochloric acid test solution to the graduation line to make 100
mL. This is used as a sample solution. Hereinafter the Method No. 1 is applied mutatis
mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of thiaminemononitrate is applied mutatis mutandis.

347

Provisional Translation from Japanese Original(52) Aluminum Hydroxide
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 33.0~36.0 % of Aluminum (Al).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product hardly dissolves in water or ethanol.
iii. This product almost dissolves in dilute hydrochloric acid or 1 mol/L of sodiumhydroxide test solution.
Confirmation test: 0.2 g (0.15~0.24 g) of this product is added with 20 mL of dilutehydrochloric acid, warmed, and filtered. The resulting filtrate gives the qualitative
reaction of aluminum salt.
Purity test:
i. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed, added with 8 mL ofhydrochloric acid and 5 mL of water, shaken up well, gently heated until boiling, andevaporated to dryness on a water bath. The residue is added with 30 mL of water,
shaken up well by heating, allowed to cool, and filtered. The filtrate is dropped with 2
mL of diluted acetic acid and 5 mL of ammonia test solution, warmed while stirring
until the solution becomes transparent, allowed to cool, added with water to make 50
mL. This is used as a sample solution. 2.0 mL of lead standard solution is added with 8
mL of hydrochloric acid and 5 mL of water, evaporated to dryness on a water bath,and subjected to the same procedure as the sample. This is used as a control solution.
When the test for heavy metal is performed using these sample and control solutions,the color of the sample solution shall not be darker than that of the control solution (10mg/kg or less).
ii. Arsenic: 0.8 g (0.75~0.84 g) of this product is weighed and added with 10 mL of dilute
sulfuric acid, gently heated while shaking until boiling, allowed to cool and filtered.
2.5 mL of filtrate is added with water to make 5 mL, which is used as a sample
solution. When the sample solution is tested for arsenic performed using device A, thecolor of the absorbing solution shall not be darker than the standard color (10 mg/kg or
less).
Assay: Approximately 2.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is added with 15 mL of hydrochloric acid, heated on a water bath for 30
minutes while shaking up, transferred to a 500 mL volumetric flask and allowed to cool,and added with water to make 500 mL. 20 mL of this solution is measured using a
volumetric pipette, added with 30 mL of 0.05 mol/L ethylenediaminetetraacetic acid348

Provisional Translation from Japanese Original

disodium solution using a volumetric pipette, added with 20 mL of pH 4.8 acetic acidammonium acetate buffer, boiled for 5 minutes, allowed to cool and added with 55 mL ofethanol, and titrated with 0.05 mol/L zinc acetate solution (indicator: 2 mL of dithizone
test solution). In this case, the end of titration is the time when the color of the solutionchanges from pale dark green to pale red. A blank test is performed in the same way.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 1.349 mg Al(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of aluminumhydroxide are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of aluminumhydroxide is applied mutatis mutandis.

(53) Taurine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 2 hours, it contains
99.0~101.0 % of taurine (C2H7NO3S).
Physical and chemical properties:
i. This product is colorless or white crystals or white crystalline powder.
ii. This product is slightly easy to dissolve in water and hardly dissolves in absoluteethanol.
iii. 1.0 g (0.95~1.04 g) of this product is dissolved in 20 mL of newly boiled and cooled
water. The pH of the resulting solution is 4.1~5.6.
Confirmation test: This product is tested by the potassium bromide disk method of theinfrared absorption spectroscopy and the spectrum of this product is compared withreference spectrum of this product. Both spectra show absorption with the similarintensity at the same wavenumber.

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Provisional Translation from Japanese Original

Transmittance(%)

Reference spectrum of this product

Wavenumber( c m −1 )Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and clear.
ii. Chloride: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution
by the chloride test method. A control solution is prepared using 0.3 mL of 0.01 mol/Lhydrochloric acid. When chloride is tested using these sample and control solutions,the turbidity of the sample solution shall not be higher than that of the control solution(0.011 % or less).
iii. Sulfate: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample solution by
the sulfate test method. A control solution is prepared using 0.4 mL of 0.005 mol/L
sulfuric acid. When sulfate is tested using these sample and control solutions, theturbidity of the sample solution shall not be higher than that of the control solution(0.010 % or less).
iv. Ammonium salt

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Provisional Translation from Japanese Original

A device described in Figure is used.
A: Distilling flask
B: Spray strip
C: Small hole
D: Condenser
E: Back-flow stopper
F: Receiver (measuring cylinder)
G: Cock(Approx. 500 mL)

H and J: Rubber stopper
K: Rubber tube

(Approx. 100 mL)

Figures are described in mm.
Operation procedures
Preparation of sample and standard solutions: 0.25 g (0.245~0.254 g) of this product isplaced in distilling flask A, dissolved with 140 mL of water, added with 2 g (1.5~2.4g) of magnesium oxide, and distilled to collect 60 mL of distillate. 20 mL of boric acidsolution (1 → 200) as an absorbing solution is put in a receiver F (measuring cylinder).
The bottom edge of the condenser is immersed in the absorbing solution and theheating temperature is adjusted to provide a distillation rate of 5~7 mL/min. The
bottom edge of the condenser is lifted from the surface of the solution and washed witha little amount of water and water is added into the receiver to make 100 mL. This isused as a sample solution. For preparation of a standard solution, 5.0 mL ofammonium standard solution is put in distilling flask A, and is hereinafter subjected tothe same procedure of preparation as the sample solution.
Tests of sample and standard solutions: 30 mL each of the sample and standard solutions
is transferred to each Nessler tube, added with 6.0 mL of phenol-sodium nitroprussidetest solution to mix them. Then, it is added with 4 mL of sodium hypochlorite-sodiumhydroxide test solution and water to make 50 mL, mixed, and allowed to stand for 60

351

Provisional Translation from Japanese Original

minutes. Each Nessler tubes are observed from above or the side using a whitebackground to compare the colors of these solutions. The color of the sample solution
shall not be darker than that of the standard solution (0.02 % or less).
Preparation of phenol-sodium nitroprusside test solution: 5 g (4.5~5.4 g) of phenol
and 25 mg (24.5~25.4 mg) of sodium nitroprusside are dissolved in water to make
500 mL. It is stored in a cool, dark place.
Preparation of sodium hypochlorite-sodium hydroxide test solution: The volume ofsodium hypochlorite test solution for the ammonia test containing 1.05 g of sodium
hypochlorite is dissolved with 15 g (14.5~15.4 g) of sodium hydroxide and water tomake 1 L. It is prepared at time of use.
v. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
vi. Analog: 1.0 g (0.95~1.04 g) of this product is dissolved in 50 mL of water to prepare asample solution. 1 mL of this sample solution is measured using a volumetric pipette,
transferred to a 50 mL volumetric flask, and added with water to the graduation line to
make 50 mL. 1 mL of this solution is measured using a volumetric pipette and addedwith water to make 10 mL. This is used as a standard solution. 5 µL each of the sample
and standard solutions is spotted on a thin layer plate prepared using silica gel for thinlayer chromatography. Then it is developed approximately 10 cm with the developing
solvent, a mixed solution of water, absolute ethanol, n-butanol, and glacial acetic acid(150:150:100:1), and the thin layer is air dried. When it is evenly sprayed withninhydrin-butanol test solution and heated at 105 °C for 5 minutes, the number of the
spots other than the main spot obtained from the sample solution is one or less, and thecolor of such spots is not darker than that obtained from the standard solution.
Loss on drying: 0.20 % or less (1 g, 105 °C, 2 hours)
Ignition residue: 0.1 % or less (1 g)Assay: This product is dried at 105 °C for 2 hours. Approximately 0.2 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved with 50 mL of water, addedwith 5 mL of formalin, and titrated with 0.1 mol/L sodium hydroxide solution(potentiometric titration). A blank test is performed in the same way and corrections are
made.
0.1 mol/L sodium hydroxide solution 1 mL = 12.52 mg C2H7NO3S

352

Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of taurine are appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of taurine isapplied mutatis mutandis.

(54) Zinc Carbonate
A. Raw material for manufacturing(a) Compositional standards
Content: This product is basic zinc carbonate. When this product is determined, it contains
57.0~60.0 % of zinc (Zn).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product dissolves in dilute hydrochloric acid or ammonia test solution and hardly
dissolves in water or ethanol.
Confirmation test:
i. A solution of this product in dilute hydrochloric acid (1 → 10) gives the qualitative
reaction of salt.
ii. This product gives the qualitative reaction i. of carbonate.
Purity test:
i. Clarity and color of solution: When 2.0 g (1.95~2.04 g) of this product is dissolved
with 10 mL of water and 30 mL of dilute sulfuric acid, the resulting solution shall becolorless and clear.
ii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is added with 10 mL of water and 10
mL of hydrochloric acid (47 → 100), dissolved while warming, allowed to cool and is
added with 3 mL of ammonia solution (4 → 5). Then, the precipitation is dissolved
with hydrochloric acid (47 → 100) and added with water to make 40 mL. This is used
as a sample solution. 15 mL of this sample solution is measured, added with 30 mL ofpotassium cyanide test solution and water to make 50 mL, added with 0.5 mL of
sodium sulfide and allowed to stand for 5 minutes. The color of the resulting solution
shall not be darker than that of a solution prepared with the way in which 5 mL of the353

Provisional Translation from Japanese Original

sample solution is added with 1.5 mL of lead standard solution, 30 mL of potassium
cyanide test solution and water to make 50 mL, added with 0.5 mL of sodium sulfide
test solution, and allowed to stand for 5 minutes (30 mg/kg or less).
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and dissolved with 5 mLof dilute hydrochloric acid to prepare a sample solution. For the sample solution, whenarsenic test is performed by the arsenic test method using device B, the color of
mercuric bromide paper shall not be darker than the standard color (5 mg/kg or less).
Loss on drying: 3.0 % or less (1 g, 105 °C, 2 hours)
Assay: Approximately 1.5 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is dissolved with 10 mL of water and 10 mL of hydrochloric acid (1 → 2),
transferred to a 100 mL volumetric flask, and added with water to the graduation line to
make 100 mL. 10 mL of this solution is measured using a volumetric pipette, added with
80 mL of water, and added with sodium hydroxide solution (1 → 50) until slight
precipitation is generated. Then it is added with 5 mL of pH 10.7 ammonia-ammonium
chloride buffer and titrated with 0.05 mol/L ethylene-diamine-tetraacetic acid disodium
solution (indicator: 0.04 g (0.035~0.044 g) of eriochrome black T-sodium chloride
indicator).
0.05 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL = 3.269 mg Zn(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of zinc carbonate areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc carbonate isapplied mutatis mutandis.

(55) Cobaltous Carbonate
A. Raw material for manufacturing(a) Compositional standards
Content: This product is basic cobaltous carbonate. When it is determined, it contains
47.0~52.0 % of cobalt (Co).
Physical and chemical properties:
i. This product is pale red or dark purple powder with no odor.

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ii. This product dissolves in dilute hydrochloric acid and hardly dissolves in water or
ethanol.
Confirmation test:
i. When 1.0 g (0.95~1.04 g) of this product is dissolved with 10 mL of dilutehydrochloric acid and added with 1 mL of α-nitroso-β-naphthol test solution, redbrown precipitation is generated.
ii. This product gives the qualitative reaction i. of carbonate.
Purity test:
i, Clarity and color of solution: When 2.0 g (1.95~2.04 g) of this product is dissolved
with 10 mL of water and 30 mL of dilute hydrochloric acid, the resulting solution shallbe reddish violet and clear.
ii. Lead: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with nitric acid (1 →150), transferred to a 20 mL volumetric flask, and added with nitric acid (1 → 150) to
the graduation line to make 20 mL. This is used as a sample solution. When lead istested by the lead test method (Method 1 of Atomic Absorption Spectrophotometry)
for the sample solution, the amount shall be 30 mg/kg or less.
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and dissolved with 5 mLof dilute hydrochloric acid to prepare a sample solution. For the sample solution, whenarsenic test is performed by the arsenic test method using device B, the color of
mercuric bromide paper shall not be darker than the standard color (5 mg/kg or less).
Loss on drying: 3.0 % or less (1 g, 70 °C, 2 hours)
Assay: Approximately 1.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is dissolved with 10 mL of water and 5 mL of hydrochloric acid (1 → 2),
transferred to a 250 mL volumetric flask, added with water to the graduation line to make
250 mL. 10 mL of this solution is measured using a volumetric pipette, added with 100
mL of water, and titrated with 0.01 mol/L ethylene-diamine-tetraacetic acid disodium
solution, while adjusting to pH 8.0 with ammonia test solution (indicator: 0.20 g(0.195~0.204 g) of murexide indicator).
0.01 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL = 0.5893 mg Co(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of cobaltous
carbonate are applied mutatis mutandis.
(b) Standard of storage method355

Provisional Translation from Japanese Original

The standard of storage method of the raw material for manufacturing of cobaltous
carbonate is applied mutatis mutandis.

(56) Sodium Bicarbonate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (silica gel) for 4 hours and determined, it
contains 99.0 % or more of sodium bicarbonate (NaHCO3).
Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor.
ii. This product is easy to slightly dissolve in water and hardly dissolves in ethanol.
iii. This product is gradually degraded in wet air.
Confirmation test: A solution of this product in water (1 → 30) gives the qualitative
reactions of sodium salt and bicarbonate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and clear.
ii. Chloride: 0.40 g (0.395~0.404 g) of this product is weighed, added with 4 mL of dilutenitric acid, heated until boiling, allowed to cool, and is added with 6 mL of dilute nitricacid and water to make 50 mL. This is used as a sample solution. When chloride istested by the chloride test method using a control solution prepared with 0.4 mL of
0.01 mol/L hydrochloric acid, the opacity of the sample solution shall not be higher
than that of the control solution (0.040 % or less).
iii. Carbonate: 1.0 g (0.95~1.04 g) of this product is weighed, added with 20 mL of newly
boiled and cooled water, extremely gently shaken at 15 °C or under to dissolve, andadded with 2.0 mL of 0.1 mol/L hydrochloric acid and 2 drops of phenolphthalein test
solution. The resulting solution shall not be red within 30 seconds.
iv. Ammonium salt: When 1.0 g (0.95~1.04 g) of this product is weighed and heated, thegenerated gas shall not change moist red litmus paper to blue.
v. Heavy metal: 4.0 g (3.95~4.04 g) of this product is weighed, added with 5 mL of waterand 4.5 mL of hydrochloric acid, and evaporated to dryness on a water bath. The
residue is dissolved with 2 mL of diluted acetic acid, 35 mL of water and a drop ofammonia test solution, and added with more water to make 50 mL. This is used as asample solution. 4.5 mL of hydrochloric acid is evaporated to dryness and added with
2 mL of diluted acetic acid, 4.0 mL of lead standard solution and water to make 50

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mL. This is used as a control solution. When these sample and control solutions aretested for heavy metal, the color of the sample solution shall not be darker than that of
the control solution (10 mg/kg or less).
vi. Arsenic: 0.70 g (0.695~0.704 g) of this product is weighed, added with 3 mL of waterand 2 mL of hydrochloric acid to prepare a sample solution. When the sample solution
is tested for arsenic by the method using device A, the color of the absorbing solution
shall not be darker than the standard color (2.8 mg/kg or less).
Assay: This product is dried in a desiccator (silica gel) for 4 hours, approximately 2.0 g of it
is weighed to the digits of 0.01 g, and the value is recorded. It is dissolved with 25 mL ofwater and titrated with 0.5 mol/L sulfuric acid. When the color of the solution changes
blue to yellow-green, it is carefully boiled, allowed to cool, and titrated until the color
changes to greenish yellow (indicator: 2 drops of bromcresol green test solution). A blanktest is performed in the same way and corrections are made.
0.5 mol/L sulfuric acid 1 mL = 84.01 mg NaHCO3(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium bicarbonate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
bicarbonate is applied mutatis mutandis.

(57) Magnesium Carbonate
A. Raw material for manufacturing(a) Compositional standards
Content: This product is hydrous basic magnesium carbonate or hydrous magnesium
orthocarbonate. When it is determined, it contains 40.0~43.5 % of magnesium oxide(MgO).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product hardly dissolves in water or ethanol.
iii. This product bubbles and dissolves in dilute hydrochloric acid.
iv. A saturated solution of this product is alkaline.

357

Provisional Translation from Japanese OriginalConfirmation test:i. 1 g (0.5~1.4 g) of this product is dissolved with 10 mL of dilute hydrochloric acid,boiled, allowed to cool, and neutralized with 1 mol/L sodium hydroxide test solution(filtered, as appropriate). The resulting solution gives the qualitative reaction ofmagnesium salt.
ii. This product gives the qualitative reaction i. of carbonate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved in 10 mLof hydrochloric acid (2 → 3) and added with 10 mL of water. The resulting solution
shall be almost clear.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, moistened with 4.0 mL ofwater, dissolved with 10 mL of dilute hydrochloric acid, and evaporated to dryness ona water bath. The residue is dissolved with 35 mL of water, 2 mL of diluted acetic
acid, and a drop of ammonia test solution, and added with more water to make 50 mL.
This is used as a sample solution. 10 mL of dilute hydrochloric acid is evaporated todryness on a water bath and added with 2 mL of diluted acetic acid, 3.0 mL of leadstandard solution and water to make 50 mL. This is used as a control solution. Whenthese sample and control solutions are tested for heavy metal, the color of the sample
solution shall not be darker than that of the control solution (30 mg/kg or less).
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed, moistened with 1.5 mL ofwater, dissolved with 3.5 mL of hydrochloric acid (1 → 4) to prepare a sample
solution. When the arsenic test is performed by the method using device A for the
sample solution, the color of absorbing solution shall not be darker than the standard
color (5 mg/kg or less).
Loss on drying: 3.0 % or less (1 g, 105 °C, 2 hours)
Assay: Approximately 1 g of this product is weighed to the digits of 0.01 g and the value isrecorded. It is dissolved with 30 mL of 0.5 mol/L sulfuric acid and excess sulfuric acid is
titrated with 1 mol/L sodium hydroxide solution (indicator: 2~3 drops of bromphenol
blue test solution). A blank test is performed in the same way.
0.5 mol/L sulfuric acid 1 mL = 20.15 mg MgO(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of magnesium
carbonate are applied mutatis mutandis.

358

Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of magnesium
carbonate is applied mutatis mutandis.

(58) Manganese Carbonate
A. Raw material for manufacturing(a) Compositional standards
Content: This is basic manganese carbonate. When it is determined, it contains 42.8~44.7 %of manganese (Mn).
Physical and chemical properties:
i. This product is light brown powder with no odor.
ii. This product dissolves in hydrochloric acid and hardly dissolves in water.
Confirmation test:
i. 1.0 g (0.95~1.04 g) of this product is dissolved with a small amount of dilutehydrochloric acid, added with 1 mol/L sodium hydroxide test solution to make itneutral. The resulting solution gives the qualitative reaction of manganese salt.
ii. This product gives the qualitative reaction i. of carbonate.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is dissolved
with 20 mL of water, 2 mL of concentrated hydrochloric acid, and a drop of hydrogen
peroxide water (3 → 100), the resulting solution shall be clear.
ii. Lead: 2.0 g (1.95~2.04 g) of this product is weighed, dissolved with 40 mL of dilutehydrochloric acid, transferred to a 100 mL volumetric flask, added with water to thegraduation line to make 100 mL. 25 mL of this solution is measured using a
volumetric pipette and this is used as a sample solution. When this sample solution istested for lead by the lead test method (dithizone method), the amount of lead shall be
20 mg/kg or less.
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and dissolved with 5 mLof dilute hydrochloric acid to prepare a sample solution. For the sample solution, when
the arsenic test is performed by the method using device B, the color of absorbing
solution shall not be darker than the standard color (5 mg/kg or less).
Loss on drying: 3.0 % or less (1 g, 70 °C, 2 hours)
Assay: Approximately 0.1 g of this product is weighed to the digits of 1 mg and the value isrecorded. It is added with 10 mL of dilute hydrochloric acid, dissolved by warming,allowed to cool, transferred to a 100 mL volumetric flask, and added with water to the359

Provisional Translation from Japanese Original

graduation line to make 100 mL. 20 mL of this solution is measured using a volumetricpipette, added with 80 mL of water, 5 mL of 0.1 mol/L magnesiumethylenediaminetetraacetic acid disodium solution, and 0.1 g (0.05~0.14 g) ofhydroxylamine hydrochloride, adjusted to pH 6.0~8.0 with dilute sodium hydroxide
solution while shaking up well, added with 1 mL of potassium cyanide solution (1 → 20)
and 2 mL of pH 10.7 ammonia-ammonium chloride buffer, and titrated with 0.01 mol/L
ethylenediaminetetraacetic acid disodium solution (indicator: 0.04 g (0.035~0.044 g) of
eriochrome black T-sodium chloride indicator). A blank test is performed in the same
way and corrections are made.
0.01 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 0.5494 mg Mn(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of manganesecarbonate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of manganesecarbonate is applied mutatis mutandis.

(59) 2-deamino-2-hydroxymethionine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 88.0 % or more of 2-deamino-2hydroxymethionine (2-hydroxy-4-metylthio butyric acid, C5H10O3S).
Physical and chemical properties:
i. This product is brown, slightly viscous liquid with a specific odor.
ii. This product is miscible in water, ether or chloroform, and extremely easy to dissolvein ethanol.
iii. The pH of this product is 1.0 or less.
Confirmation test:
i. 25 mg (24.5~25.4 mg) of this product is added with 1 mL of sulfuric acid saturated
with anhydrous copper sulfate. The resulting solution is yellow.
ii. 5 mg (4.5~5.4 mg) of this product is dissolved with 5 mL of water, added with 2 mLof 1 mol/L sodium hydroxide test solution, shaken up well, added with 0.3 mL of360

Provisional Translation from Japanese Original

sodium nitroprusside test solution, shaken up well again, allowed to stand at 35~40 °C
for 10 minutes, cooled with ice for 2 minutes, added with 2 mL of dilute hydrochloric
acid and mixed. The resulting solution is red.
iii. 25 mg (24.5~25.4 mg) of this product is added with 2 mL of a solution in which 1 mg
(0.5~1.4 mg) of 2,7-dioxynaphthalene is dissolved in 10 mL of sulfuric acid, and iswarmed on a water bath at 90 °C for 2 minutes. The resulting solution emits yellowgreen fluorescence.
Purity test:
i. Sulfate: 0.025 g (0.0245~0.0254 g) of this product is weighed to prepare a sample
solution by the sulfate test method. A control solution is prepared using 0.5 mL of
0.005 mol/L sulfate. When sulfate is tested using these sample and control solutions,the turbidity of the sample solution shall not be higher than that of the control solution(0.96 % or less).
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 1 g (0.5~1.4 g) of this product is weighed and a sample solution is preparedby Method No. 1 of the arsenic test method. The arsenic test is performed by themethod using device A. The color of absorbing solution shall not be darker than thestandard color (2 mg/kg or less).
Content of water: 10.0~12.5 % (Direct titration).
Assay: Approximately 0.20 g of this product is weighed to the digits of 1 mg and the value
is recorded. It is dissolved with 50 mL of a mixed solution of glacial acetic acid,hydrochloric acid, and water (40:10:3), and titrated with 0.05 mol/L bromine solution(Potentiometric titration).
A platinum electrode and a double junction electrode is used as a indicating electrode anda reference electrode, respectively.
0.05 mol/L bromine solution 1 mL = 7.510 mg C5H10O3S(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of 2-deamino-2hydroxymethionine are applied mutatis mutandis.

361

Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of 2-deamino-2hydroxymethionine is applied mutatis mutandis.
(c) Standard of labeling
The following words shall be described on the immediate container or the immediatewrapper of this product.
Precaution for use: This feed additive requires careful handling due to its low pH.

(60) Zn bis (2-hydroxy-4-metylthio butyrate)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 80.0~84.0 % of 2-deamino-2hydroxymethionine (2-hydroxy-4-metylthio butyric acid, C5H10O3S) and 16.0~20.0 % ofzinc.
Physical and chemical properties: This product is gray powder with a specific odor.
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is dissolved with 5 mL of water, added with 2 mL of
1 mol/L sodium hydroxide test solution, shaken up well, added with 0.3 mL of sodium
nitroprusside test solution, shaken up well again, allowed to stand at 35~40 °C for 10
minutes, cooled with ice for 2 minutes, added with 2 mL of dilute hydrochloric acidand mixed. The resulting solution is red.
ii. 0.5 g (0.45~0.54 g) of this product is weighed, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and warmed in a water bath at 60 °C for 3 minutes
while gently shaking. It is allowed to cool, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and shaken up. 10 mL of this solution is measured,
added with 10 mL of trichloroacetic acid solution (1 → 10), shaken up, and allowed tostand for 10 minutes. This solution is filtered with filter paper and the filtrate is used asa sample solution. 1 mL of the sample solution is measured, and added with 1~2 dropsof pyridine and 1 mL of potassium thiocyanate test solution. White precipitation isformed in the resulting solution.
iii. When the infrared absorption spectrum of this product is measured using the ATR
method of the infrared absorption spectroscopy, absorptions are observed atwavelengths around 1,625 cm−1, 1,577 cm−1, and 1,370cm−1.

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Provisional Translation from Japanese Original

Purity test:i. Lead: 0.67 g (0.665~0.674 g) of this product is weighed, added with 3 mL of nitric acidand 5 mL of perchloric acid, evaporated to dryness, allowed to cool, added with 5 mLof dilute hydrochloric acid, and warmed on a water bath to dissolve. It is allowed tocool, added with 5 mL of water, mixed, and filtered using filter paper. The residue iswashed with 5 mL of water. The filtrate and the washings are mixed, transferred to a
25 mL volumetric flask, and added with water to the graduation line to make 25 mL.
This is used as a sample solution. Separately, 4 mL of lead standard solution foratomic absorption is measured using a volumetric pipette and put in a 50 mL
volumetric flask, and added with water up to the capacity mark to make 50 mL. 10 mLof this solution is measured using a volumetric pipette and put in a 25 mL volumetricflask, and added with 5 mL of dilute hydrochloric acid and water up to the capacity
mark to make 25 mL. This is used as a standard solution. When the sample andstandard solutions are measured by the atomic absorption spectrophotometry (flametype) under the following conditions, the absorbance of the sample solution shall belower than that of the standard solution (30 μg/g or less).
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Lead hollow cathode lamp
Wavelength: 217.0 nm
ii. Arsenic: 0.20 g (0.195~0.204 g) of this product is weighed and the sample solution isprepared using method No. 3 of the arsenic test method. The arsenic test is performedin the method using device A. The color of the absorbing solution shall not be darker
than the standard color (10 μg/g or less).
Assay:
i. 2-deamino-2-hydroxymethionine (2-hydroxy-4-metylthio butyric acid): Approximately
0.5 g of this product is weighed to the digit of 0.0001 g and the value is recorded. It isdissolved with 50 mL of a mixed solution of glacial acetic acid, water, andhydrochloric acid (50:10:3), and titrated with 0.05 mol/L bromine solution(potentiometric titration).
0.05 mol/L bromine solution 1 mL = 7.510 mg C5H10O3S
ii. Zinc: Approximately 0.2 g of this product is measured to the digit of 0.001 g and thevalue is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitric acid and10 mL of perchloric acid and heated until the amount of the residual liquid becomesapproximately 2 mL. It is allowed to cool, added with 2 mL of hydrochloric acid and363

Provisional Translation from Japanese Original

heated on a water bath to dissolve. It is allowed to cool, transferred to a 100 mL
volumetric flask, and added with water up to the capacity mark to make 100 mL. Thissolution is filtered using filter paper. 10 mL of the first filtrate is discarded, 10 mL ofthe next filtrate is measured using a volumetric pipette and put in a 100 mL volumetricflask, and added with water up to the capacity mark to make 100 mL. Then, 5 mL ofthis solution is measured using a volumetric pipette and put in a 100 mL volumetricflask, and added with water up to the capacity mark to make 100 mL. This is used as asample solution. Separately, 5 mL of nitric acid and 10 mL of perchloric acid areplaced in a Kjeldahl flask and heated until the amount of the residual liquid becomesapproximately 2 mL. It is allowed to cool, added with 2 mL of hydrochloric acid,transferred to a 100 mL volumetric flask, and added with water up to the capacity
mark to make 100 mL. 10 mL of this solution is measured using a volumetric pipetteand put in a 100 mL volumetric flask, and added with water up to the capacity mark to
make 100 mL. This is used as a blank test stock solution. Then, 5 mL of this solution ismeasured using a volumetric pipette and put in a 100 mL volumetric flask, and addedwith water up to the capacity mark to make 100 mL. This is used as a blank test
solution. Separately, 5, 10, 15 and 20 mL each of zinc standard solutions is measured
using a volumetric pipette and put in a 100 mL volumetric flask, 5 mL of the blank test
stock solution is added using a volumetric pipette, and added with water up to thecapacity mark to make 100 mL. The resulting solutions are called the standard solution
1, 2, 3 and 4, respectively. The sample solution, the standard solutions 1, 2, 3 and 4,
and the blank test solution are determined by the atomic absorption spectrophotometry(flame type) under the following conditions. A calibration curve is prepared from theabsorbances of the standard solutions 1, 2, 3 and 4, and the blank test solution, and thecontent of zinc in the sample is derived.
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Zinc hollow cathode lamp
Wavelength: 213.9 nm
Content of zinc (%) =Concentration of zine in the sample solution derived from the calibration curve (μg/mL)
Collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a sealed container.

364

× 2000Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of Zn bis (2-hydroxy4-methylthio butyrate) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Zn bis (2hydroxy-4-methylthio butyrate) is applied mutatis mutandis.

(61) Cu bis (2-hydroxy-4-metylthio butyrate)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 78.0~83.0 % of 2-deamino-2hydroxymethionine (2-hydroxy-4-metylthio butyric acid, C5H10O3S) and 15.0~20.0 % ofcopper.
Physical and chemical properties: This product is gray to green powder with a specific odor.
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is dissolved with 5 mL of water, added with 2 mL of
1 mol/L sodium hydroxide test solution, shaken up well, added with 0.3 mL of sodium
nitroprusside test solution, shaken up well again, allowed to stand at 35~40 °C for 10
minutes, cooled with ice for 2 minutes, added with 2 mL of dilute hydrochloric acidand mixed. The resulting solution is red and precipitation is generated.
ii. 0.5 g (0.45~0.54 g) of this product is weighed, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and warmed in a water bath at 60 °C for 3 minutes
while gently shaking. It is allowed to cool, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and shaken up. 10 mL of this solution is measured,
added with 10 mL of trichloroacetic acid solution (1 → 10), shaken up, and allowed tostand for 10 minutes. This solution is filtered with filter paper and the filtrate is used asa sample solution. 1 mL of the sample solution is measured, and added with 1 mL ofpotassium ferrocyanide test solution. Red-brown precipitation is formed in the
resulting solution.
iii. When the infrared absorption spectrum of this product is measured using the ATR
method of the infrared absorption spectroscopy, absorptions are observed atwavelengths around 1,683 cm−1, 1,591 cm−1, 1,552 cm−1, and 1,416cm−1.

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Provisional Translation from Japanese Original

Purity test:i. Lead: 0.67 g (0.665~0.674 g) of this product is weighed, added with 3 mL of nitric acidand 5 mL of perchloric acid, evaporated to dryness, allowed to cool, added with 5 mLof dilute hydrochloric acid, and warmed on a water bath to dissolve. It is allowed tocool, added with 5 mL of water, mixed, and filtered using filter paper. The residue iswashed with 5 mL of water. The filtrate and the washings are mixed, transferred to a
25 mL volumetric flask, and added with water to the graduation line to make 25 mL.
This is used as a sample solution. Separately, 4 mL of lead standard solution foratomic absorption is measured using a volumetric pipette and put in a 50 mL
volumetric flask, and added with water up to the capacity mark to make 50 mL. 10 mLof this solution is measured using a volumetric pipette and put in a 25 mL volumetricflask, and added with 5 mL of dilute hydrochloric acid and water up to the capacity
mark to make 25 mL. This is used as a standard solution. When the sample andstandard solutions are measured by the atomic absorption spectrophotometry (flametype) under the following conditions, the absorbance of the sample solution shall belower than that of the standard solution (30 μg/g or less).
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Lead hollow cathode lamp
Wavelength: 217.0 nm
ii. Arsenic: 0.20 g (0.195~0.204 g) of this product is weighed and the sample solution isprepared using method No. 3 of the arsenic test method. The arsenic test is performedin the method using device A. The color of the absorbing solution shall not be darker
than the standard color (10 μg/g or less)
Assay:
i. 2-deamino-2-hydroxymethionine (2-hydroxy-4-metylthio butyric acid): Approximately
0.5 g of this product is weighed to the digit of 0.0001 g and the value is recorded. It isdissolved with 50 mL of a mixed solution of glacial acetic acid, water, andhydrochloric acid (50:10:3), and titrated with 0.05 mol/L bromine solution(potentiometric titration).
0.05 mol/L bromine solution 1 mL = 7.510 mg C5H10O3S
ii. Copper: Approximately 0.2 g of this product is measured to the digit of 0.001 g andthe value is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitric acidand 10 mL of perchloric acid and heated until the amount of the residual liquid
becomes approximately 2 mL. It is allowed to cool, added with 2 mL of hydrochloric

366

Provisional Translation from Japanese Originalacid and heated on a water bath to dissolve. It is allowed to cool, transferred to a 100mL volumetric flask, and added with water up to the capacity mark to make 100 mL.
This solution is filtered using filter paper. 10 mL of the first filtrate is discarded, 10
mL of the next filtrate is measured using a volumetric pipette and put in a 100 mL
volumetric flask, and added with water up to the capacity mark to make 100 mL. Then,
5 mL of this solution is measured using a volumetric pipette and put in a 100 mL
volumetric flask, and added with water up to the capacity mark to make 100 mL. Thisis used as a sample solution. Separately, 5 mL of nitric acid and 10 mL of perchloric
acid are placed in a Kjeldahl flask and heated until the amount of the residual liquid
becomes approximately 2 mL. It is allowed to cool, transferred to a 100 mL volumetricflask, added with 2 mL of hydrochloric acid, and added with water up to the capacity
mark to make 100 mL. 10 mL of this solution is measured using a volumetric pipetteand put in a 100 mL volumetric flask, and added with water up to the capacity mark to
make 100 mL. This is used as a blank test stock solution. Then, 5 mL of this solution ismeasured using a volumetric pipette and put in a 100 mL volumetric flask, and addedwith water up to the capacity mark to make 100 mL. This is used as a blank test
solution. Separately, 2, 4, 6 and 8 mL each of copper standard solutions is measured
using a volumetric pipette and put in a 100 mL volumetric flask, 5 mL of the blank test
stock solution is added using a volumetric pipette, and added with water up to thecapacity mark to make 100 mL. The resulting solutions are called the standard solution
1, 2, 3 and 4, respectively. The sample solution, the standard solutions 1, 2, 3 and 4,
and the blank test solution are determined by the atomic absorption spectrophotometry(flame type) under the following conditions. A calibration curve is prepared from theabsorbances of the standard solutions 1, 2, 3 and 4, and the blank test solution, and thecontent of copper in the sample is derived.
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Copper hollow cathode lamp
Wavelength: 324.7 nm
Content of copper (%) =Concentration of copper in the sample solution derived from the calibration curve (μg/mL)
×2000
Collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a sealed container.

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Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of Cu bis (2-hydroxy4-methylthio butyrate) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cu bis (2hydroxy-4-methylthio butyrate) is applied mutatis mutandis.

(62) Mn bis (2-hydroxy-4-metylthio butyrate)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 76.0~82.0 % of 2-deamino-2hydroxymethionine (2-hydroxy-4-metylthio butyric acid, C5H10O3S) and 13.0~19.0 % ofmanganese.
Physical and chemical properties: This product is gray to brown powder with a specificodor.
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is dissolved with 5 mL of water, added with 2 mL of
1 mol/L sodium hydroxide test solution, shaken up well, added with 0.3 mL of sodium
nitroprusside test solution, shaken up well again, allowed to stand at 35~40 °C for 10
minutes, cooled with ice for 2 minutes, added with 2 mL of dilute hydrochloric acidand mixed. The resulting solution is red.
ii. 0.5 g (0.45~0.54 g) of this product is weighed, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and warmed in a water bath at 60 °C for 3 minutes
while gently shaking. It is allowed to cool, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and shaken up. 10 mL of this solution is measured,
added with 10 mL of trichloroacetic acid solution (1 → 10), shaken up, and allowed tostand for 10 minutes. This solution is filtered with filter paper and the filtrate is used asa sample solution. 1 mL of the sample solution is measured, and added with ammonia
test solution and 5 mL of silver nitrate test solution. White precipitation is formed in
the resulting solution, and the generated precipitation gradually turns black.
iii. When the infrared absorption spectrum of this product is measured using the ATR
method of the infrared absorption spectroscopy, absorptions are observed atwavelengths around 1,627 cm−1, 1,574 cm−1, and 1,435cm−1.

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Purity test:i. Lead: 0.67 g (0.665~0.674 g) of this product is weighed, added with 3 mL of nitric acidand 5 mL of perchloric acid, evaporated to dryness, allowed to cool, added with 5 mLof dilute hydrochloric acid, and warmed on a water bath to dissolve. It is allowed tocool, added with 5 mL of water, mixed, and filtered using filter paper. The residue iswashed with 5 mL of water. The filtrate and the washings are mixed, transferred to a
25 mL volumetric flask, and added with water to the graduation line to make 25 mL.
This is used as a sample solution. Separately, 4 mL of lead standard solution foratomic absorption is measured using a volumetric pipette and put in a 50 mL
volumetric flask, and added with water up to the capacity mark to make 50 mL. 10 mLof this solution is measured using a volumetric pipette and put in a 25 mL volumetricflask, and added with 5 mL of dilute hydrochloric acid and water up to the capacity
mark to make 25 mL. This is used as a standard solution. When the sample andstandard solutions are measured by the atomic absorption spectrophotometry (flametype) under the following conditions, the absorbance of the sample solution shall belower than that of the standard solution (30 μg/g or less).
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Lead hollow cathode lamp
Wavelength: 217.0 nm
ii. Arsenic: 0.10 g (0.095~0.104 g) of this product is weighed and the sample solution isprepared using method No. 3 of the arsenic test method. The arsenic test is performedin the method using device A. The color of the absorbing solution shall not be darker
than the standard color (20 μg/g or less)
Assay:
i. 2-deamino-2-hydroxymethionine (2-hydroxy-4-metylthio butyric acid): Approximately
0.5 g of this product is weighed to the digit of 0.0001 g and the value is recorded. It isdissolved with 50 mL of a mixed solution of glacial acetic acid, water, andhydrochloric acid (50:10:3), and titrated with 0.05 mol/L bromine solution(potentiometric titration).
0.05 mol/L bromine solution 1 mL = 7.510 mg C5H10O3S
ii. Manganese: Approximately 0.2 g of this product is measured to the digit of 0.001 g
and the value is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitricacid and 10 mL of perchloric acid and heated until the amount of the residual liquid
becomes approximately 2 mL. It is allowed to cool, added with 2 mL of hydrochloric

369

Provisional Translation from Japanese Originalacid and heated on a water bath to dissolve. It is allowed to cool, transferred to a 100mL volumetric flask, and added with water up to the capacity mark to make 100 mL.
This solution is filtered using filter paper. 10 mL of the first filtrate is discarded, 10
mL of the next filtrate is measured using a volumetric pipette and put in a 100 mL
volumetric flask, and added with water up to the capacity mark to make 100 mL. Then,
5 mL of this solution is measured using a volumetric pipette and put in a 100 mL
volumetric flask, and added with water up to the capacity mark to make 100 mL. Thisis used as a sample solution. Separately, 5 mL of nitric acid and 10 mL of perchloric
acid are placed in a Kjeldahl flask and heated until the amount of the residual liquid
becomes approximately 2 mL. It is allowed to cool, transferred to a 100 mL volumetricflask, added with 2 mL of hydrochloric acid, and added with water up to the capacity
mark to make 100 mL. 10 mL of this solution is measured using a volumetric pipetteand put in a 100 mL volumetric flask, and added with water up to the capacity mark to
make 100 mL. This is used as a blank test stock solution. Then, 5 mL of this solution ismeasured using a volumetric pipette and put in a 100 mL volumetric flask, and addedwith water up to the capacity mark to make 100 mL. This is used as a blank test
solution. Separately, 5, 10, 15 and 20 mL each of manganese standard solutions ismeasured using a volumetric pipette and put in a 100 mL volumetric flask, 5 mL of theblank test stock solution is added using a volumetric pipette, and added with water up
to the capacity mark to make 100 mL. The resulting solutions are called the standard
solution 1, 2, 3 and 4, respectively. The sample solution, the standard solutions 1, 2, 3
and 4, and the blank test solution are determined by the atomic absorption
spectrophotometry (flame type) under the following conditions. A calibration curve isprepared from the absorbances of the standard solutions 1, 2, 3 and 4, and the blanktest solution, and the content of manganese in the sample is derived.
Gas to be used:
Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Manganese hollow cathode lamp
Wavelength: 279.5 nm
Content of manganse (%) =Concentration of manganese in the sample solution derived from the calibration curve (μg/mL)
×2000
Collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a sealed container.

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Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of Mn bis (2-hydroxy4-methylthio butyrate) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Mn bis (2hydroxy-4-methylthio butyrate) is applied mutatis mutandis.

(63) DL-Tryptophan
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.5 % or more of DL-tryptophan (C11H12N2O2).
Physical and chemical properties:
i. This product is white to pale yellow powder with no odor or a slight specific odor.
ii. This product is hard to dissolve in water, extremely hard to dissolve in ethanol andhardly dissolves in ether.
iii. This product dissolves in dilute hydrochloric acid or 1 mol/L sodium hydroxide test
solution.
iv. The pH of a solution (1 → 500) of this product is 5.5~7.0.
Confirmation test:
i. A solution of this product in water (1 → 500) does not have an optical rotation.
ii. 0.1 g (0.05~0.14 g) of this product is added with 50 mL of water, dissolved by heating
and is allowed to cool. When 10 mL of the resulting solution is added with 5 mL of pdimethylaminobenzaldehyde-ferric chloride test solution and 2 mL of dilutehydrochloric acid, heated in a water bath for 5 minutes, it is reddish violet to blueviolet.
Purity test:
i. Chloride: 0.09 g (0.085~0.094 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.5 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.20 % or less).

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ii. Ammonium salt: The purity test iii. for the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis. In this case, “0.1 g” shall be replaced by“0.05 g” (0.04 % or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 1.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 0.5 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours. Approximately 0.3 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (indicator: 10 drops of α-naphtholbenzein test solution). In thiscase, the end of titration is the time when the color of the solution changes from brown togreen. A blank test is performed in the same way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 20.42 mg C11H12N2O2(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of DL-tryptophan are
applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of DL-tryptophan
is applied mutatis mutandis.

(64) L-Tryptophan
L-Tryptophan (Part 1)
A. Raw material for manufacturing(a) Compositional standards372

Provisional Translation from Japanese OriginalContent: When this product is determined after dried at 105 °C for 3 hours, it contains
97.0 % or more of L-tryptophan (C11H12N2O2).
Physical and chemical properties:
i. This product is white to pale yellow powder with no odor or a slight specific odor.
ii. This product is hard to dissolve in water, extremely hard to dissolve in ethanol, andhardly dissolves in ether.
iii. This product dissolves in dilute hydrochloric acid or 1 mol/L sodium hydroxide test
solution.
iv. The pH of a solution (1 → 500) of this product is 4.5~7.0.
Confirmation test:
i. A solution of this product in water (1 → 100) shows levorotation, which changes todextrorotation when the solution is added with sodium hydroxide solution (1 → 5) tomake it alkaline.
ii. The confirmation test ii, of the raw material for manufacturing of DL-tryptophan isapplied mutatis mutandis.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours. Approximately 0.5 g of it
is weighed to the digits of 0.001 g and the value is recorded. It is added withapproximately 40 mL of water, dissolved by heating, allowed to cool, added withwater to make 50 mL, and if necessary filtered with a membrane filter (0.45 µm). The
rotation of this solution shall be [α]20
D = -28.4 ~ -33.0°.

ii. Chloride: The purity test i. of the raw material for manufacturing of DL-tryptophan isapplied mutatis mutandis (0.20 % or less).
iii. Ammonium salt: The purity test ii. of the raw material for manufacturing of DLtryptophan is applied mutatis mutandis (0.2 % or less).
iv. Heavy metal: The purity test iii. of the raw material for manufacturing of DLtryptophan is applied mutatis mutandis (20 mg/kg or less).
v. Arsenic: The purity test iv. of the raw material for manufacturing of DL-tryptophan isapplied mutatis mutandis (2 mg/kg or less).
Loss on drying: 1.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 1.0 % or less (1 g)Assay: The assay of the raw material for manufacturing of DL-tryptophan is applied mutatis
mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

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Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-tryptophan (part1) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-tryptophan(part 1) is applied mutatis mutandis.
L-Tryptophan (Part 2)
A. Raw material for manufacturing(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-tryptophan (part1) are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the L-tryptophan producing strain of Escherichia coli is cultured.
After the cultivation the bacterial cells are heat-treated to separate L-tryptophan, and dried.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-tryptophan(part 1) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-tryptophan (part2) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-tryptophan(part 2) is applied mutatis mutandis.

(65) L-Threonine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.5 % or more of L-threonine (C4H9NO3).
Physical and chemical properties:
i. This product is white to light yellowish brown crystals or crystalline powder with noodor or a slight specific odor.

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Provisional Translation from Japanese Original

ii. The pH of a solution (1 → 20) of this product is 5.0~6.5.
Confirmation test:
i. 5 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes. The resulting solution is reddish
violet to purple.
ii. When 5 mL of a solution of this product in water (1 → 10) is added with 5 mL ofsaturated potassium periodate and heated, a gas with an ammonia odor is produced.
This gas changes moist red litmus paper to blue.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours. Approximately 3 g of it isweighed to the digits of 0.01 g and the value is recorded. It is dissolved in water tomake 50 mL, and filtered as appropriate. The rotation of this solution shall be[α]20
D = -26.0 ~ -29.0°.

ii. Chloride: 0.09 g (0.085~0.094 g) of this product is weighed to prepare a sample
solution by the chloride test method. A control solution is prepared using 0.5 mL of
0.01 mol/L hydrochloric acid. When chloride is tested using these samples and control
solutions, the turbidity of the sample solution shall not be higher than that of thecontrol solution (0.20 % or less).
iii. Ammonium salt: The purity test iii. for the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis. In this case, “0.1 g” shall be replaced by“0.02 g” (0.10 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 0.5 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours. Approximately 0.2 g of it is weighed tothe digits of 0.001 g and the value is recorded. It is dissolved in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (potentiometric titration). The end of titration using an indicator (1375Provisional Translation from Japanese Original

mL of crystal violet-glacial acetic acid test solution) is the time when the color of thesolution changes from purple through blue to green. A blank test is performed in the same
way and corrections are made.
0.1 mol/L perchloric acid 1 mL = 11.91 mg C4H9NO3(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L- threonine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L- threonine isapplied mutatis mutandis.

(66) Ferrous DL-Threonine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (reduced pressure, silica gel) for 4 hours
and determined, it contains 58.0~67.0 % of DL-threonine (C4H9NO3) and 13.6~15.7 % offerrous iron [Fe (Ⅱ)].
Physical and chemical properties:
i. This product is pale yellow to light brown powder with a slight specific odor.
ii. This product dissolves in dilute hydrochloric acid, and hardly dissolves in water,
ethanol or acetone.
Confirmation test:
i. A solution of 0.1 g (0.05~0.14 g) of this product in 10 mL of dilute hydrochloric acidgives the qualitative reaction of ferrous salt.
ii. 0.1 g (0.05~0.14 g) of this product is added with 5 ml of 1 mol/L hydrochloric acidtest solution and boiled until aldehyde odor is eliminated. It is allowed to cool, addedwith ammonia test solution to make it alkaline, and filtered. Filter paper is immersed in
the filtrate and then air-dried. When it is evenly sprayed with a solution of ninhydrin inwater-saturated n-butanol (1 → 500) and heated at 100 °C for 10 minutes, it is purple.
Purity test:i. Lead: 1.0 g (0.95~1.04 g) of this product is weighed and added with 20 mL of waterand 5 mL of nitric acid (1 → 3) and gently boiled for 5 minutes. It is allowed to cool,376

Provisional Translation from Japanese Originaltransferred to a 50 mL volumetric flask and added with water to make 50 mL. 25 mLof this solution is measured using a volumetric pipette to prepare a sample solution.
When this sample solution is tested for lead by the lead test method (dithizone
method), the amount of lead shall be 20 mg/kg or less.
ii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and dissolved with 2 mL of
dilute hydrochloric acid and 3 mL of water to prepare a sample solution. When thissample solution is tested for arsenic by the method using device A, the color of theabsorbing solution shall not be darker than the standard color (5 mg/kg or less).
Loss on drying: 1.0 % or less (0.5 g, silica gel, reduced pressure, 4 hours)
Assay:
i. DL-threonine: This product is dried in a desiccator (reduced pressure, silica gel) for 4
hours, approximately 0.3 g of it is weighed to the digits of 0.001 g, and the value isrecorded. It is added with 50 mL of water and heated on a water bath forapproximately 3 hours until the specific odor is eliminated. It is allowed to cool, addedwith 10 mL of formalin previously neutralized with 0.1 mol/L sodium hydroxide
solution using phenolphthalein test solution by a volumetric pipette, and titrated with
0.1 mol/L sodium hydroxide solution (potentiometric titration). A blank test is
performed in the same manner and corrections are made.
0.1 mol/L sodium hydroxide solution 1 mL = 11.91 mg C4H9NO3
ii. Ferrous iron: This product is dried in a desiccator (reduced pressure, silica gel) for 4
hours, approximately 0.6 g of it is weighed to the digits of 0.001 g, and the value isrecorded. It is dissolved with 10 mL of 0.5 mol/L sulfuric acid, and titrated with 0.1
mol/L ceric ammonium sulfate solution (indicator: 3 drops of o-phenanthroline test
solution). A blank test is performed in the same manner and corrections are made.
0.1 mol/L ceric ammonium sulfate solution 1 mL = 5.585 mg Fe(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of ferrous DLthreonine are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ferrous DLthreonine is applied mutatis mutandis.

377

Provisional Translation from Japanese Original(67) Nicotinic Acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 1 hour, it contains
99.0 % or more of nicotinic acid (C6H5NO2).
Physical and chemical properties:
i. This product is white to pale yellow white crystals or crystalline powder with no odor
or a slight specific odor and with a slight sour taste.
ii. This product is slightly hard to dissolve in water, hard to dissolve in ethanol, andextremely hard to dissolve in ether.
iii. This product dissolves in 1 mol/L sodium hydroxide test solution or sodium carbonate
test solution.
iv. The pH of a solution (1 → 100) of this product is 3.0~4.0.
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is mixed with 0.01 g (0.005~0.014 g) of 2,4dinitrochlorobenzene, melted by gentle heating for 5~6 seconds, allowed to cool, andadded with 4 mL of potassium hydroxide-ethanol test solution. The resulting solution
is dark red.
ii. 2 mL of a solution of this product in water (1 → 1,000) is added with 1 mL of
cyanogen bromide test solution and 1 mL of aniline solution (1 → 40). The resulting
solution is yellow.
iii. 0.02 g (0.015~0.024 g) of this product is dissolved with water to make 1 L. In themeasurement of the absorption spectrum of this solution, the absorption maximum andminimum are at the wavelengths of 261~263 nm and 235~239 nm, respectively. Whenthe absorbances at the absorption maximum and minimum are A1 and A2, respectively,
A2/A1 is 0.35~0.39.
Purity test:
i. Melting point: The melting point of this product shall be 234~238 °C
ii. Chloride: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by the chloride test method. A control solution is prepared using 0.30 mL of 0.01
mol/L hydrochloric acid. When chloride is tested using these sample and control
solutions, the opacity of the sample solution shall not be higher than that of the control
solution (0.021 % or less).
iii. Sulfate: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with 3 mL of dilutehydrochloric acid and water to make 50 mL. The resulting solution is used as a sample
solution. 0.40 mL of 0.005 mol/L sulfuric acid is added with 3 mL of dilute378

Provisional Translation from Japanese Originalhydrochloric acid and water to make 50 mL. This is used as a control solution. Whensulfate is tested using these solutions, the opacity of the sample solution shall not behigher than that of the control solution (0.019 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 0.5 % or less (1 g, 105 °C, 1 hour)
Ignition residue: 0.10 % or less (1 g)Assay: This product is dried at 105 °C for 1 hour, approximately 0.3 g of it is weighed to thedigits of 0.001 g, and the value is recorded. It is dissolved with 50 mL of water andtitrated with 0.1 mol/L sodium hydroxide solution (indicator: 5 drops of phenolphthaleintest solution).
0.1 mol/L sodium hydroxide solution 1 mL = 12.31 mg C6H5NO2(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of nicotinic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of nicotinic acid isapplied mutatis mutandis.

(68) Nicotinamide
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (reduced pressure, silica gel) for 4 hours
and determined, it contains 98.5 % or more of nicotinamide (C6H6N2O).
Physical and chemical properties:
i. This product is white to pale yellow white crystals or crystalline powder with no odor
or a slight specific odor and with a bitter taste.
ii. This product is easy to dissolve in water, ethanol or glycerin, and hard to dissolve in
ether.

379

Provisional Translation from Japanese Originaliii. The melting point of this product is 124~131 °C.
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is mixed with 0.01 g (0.005~0.014 g) of 2,4dinitrochlorobenzene, melted by gentle heating for 5~6 seconds, allowed to cool, andadded with 4 mL of potassium hydroxide-ethanol test solution. The resulting solution
is red.
ii. 1 mg (0.5~1.4 mg) of this product is dissolved with 100 mL of pH 7.0 phosphatebuffer. 2 mL of this solution is added with 1 mL of cyanogen bromide test solution,
heated at 80 °C for 7 minutes, rapidly cooled, added with 5 mL of 1 mol/L sodiumhydroxide solution, and allowed to stand for 30 minutes. When the resulting solution isobserved under ultraviolet light at a wavelength of 365 nm, it emits blue fluorescence.
iii. When 0.02 g (0.015~0.024 g) of this product is added with 5 mL of 1 mol/L sodiumhydroxide solution and carefully boiled, the produced gas changes moist red litmus
paper to blue.
iv. 0.02 g (0.015~0.024 g) of this product is dissolved with water to make 1 L. In themeasurement of the absorption spectrum of this solution, the absorption maximum andminimum are at the wavelengths of 261~263 nm and 243~247 nm, respectively. Whenthe absorbances at the absorption maximum and minimum are A1 and A2, respectively,
A2/A1 is 0.63~0.67.
Purity test:
i. pH: The pH of a solution of this product in water (1 → 10) shall be 5.0~7.5.
ii. Chloride: 0.5 g (0.45~0.54 g) of this product is weighed to prepare a sample solution
by the chloride test method. A control solution is prepared using 0.30 mL of 0.01
mol/L hydrochloric acid. When chloride is tested using these sample and control
solutions, the opacity of the sample solution shall not be higher than that of the control
solution (0.021 % or less).
iii. Sulfate: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample solution by
the sulfate test method. A control solution is prepared using 0.40 mL of 0.005 mol/L
sulfuric acid. When sulfate is tested using these sample and control solutions, theopacity of the sample solution shall not be higher than that of the control solution(0.019 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 3.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (30 mg/kg or less).

380

Provisional Translation from Japanese Originalv. Readily carbonizable substances: When 0.2 g (0.15~0.24 g) of this product is weighed
and conducted readily carbonizable substances test, its color shall not be darker thanthat of the control solution A.
Loss on drying: 0.5 % or less (1 g, reduced pressure, silica gel, 4 hours)
Ignition residue: 0.80 % or less (1 g)Assay: This product is dried in a desiccator (reduced pressure, silica gel) for 4 hours,
approximately 0.3 g of it is weighed to the digits of 0.001 g, and the value is recorded. It
is added with 20 mL of glacial acetic acid for nonaqueous titration, and heated if
necessary to dissolve, allowed to cool, added with 100 mL of benzene, and titrated with
0.1 mol/L perchloric acid (indicator: 2 drops of methylrosanilinium chloride test
solution).

In this case, the end of titration is the time when the color of the solutionchanges from purple through blue to blue-green. A blank test is performed in the samemanner and corrections are made.
0.1 mol/L perchloric acid 1 mL = 12.21 mg C6H6N2O(b) Standard of storage method
It shall be stored in an airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of nicotinamide areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of nicotinamide isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof nicotinamide and excipient.
Content: When this product is determined, it contains nicotinamide (C6H6N2O)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 5 mg of
the raw material for manufacturing of nicotinamide is mixed with 0.01 g (0.005~0.014g) of 2,4-dinitrochlorobenzene, and the confirmation test i. of the raw material for
manufacturing of nicotinamide is hereinafter applied mutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 1 mg of
the raw material for manufacturing of nicotinamide is added with 100 mL of pH 7.0

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Provisional Translation from Japanese Original

phosphate buffer, allowed to stand for 15 minutes with occasional shaking, andfiltered. 2 mL of this solution is added with 1 mL of cyanogen bromide test solution
and the confirmation test ii. of the raw material for manufacturing of nicotinamide ishereinafter applied mutatis mutandis.
iii. According to the amount of this product on the label, the amount containing 0.02 g of
the raw material for manufacturing of nicotinamide is added with 5 mL of 1 mol/L
sodium hydroxide solution, and the confirmation test iii. of the raw material for
manufacturing of nicotinamide is hereinafter applied mutatis mutandis.
iv. According to the amount of this product on the label, the amount containing 0.02 g of
the raw material for manufacturing of nicotinamide is weighed, added with 1 L ofwater, allowed to stand for 15 minutes with occasional shaking, and filtered. To theresulting solution, the confirmation test iv. of the raw material for manufacturing of
nicotinamide is hereinafter applied.
Assay: This product is determined using the device for the nitrogen determination method(Semimicro-Kjeldahl Method).
The amount of this product containing approximately 0.03 g of nicotinamide (C6H6N2O)
is weighed to three significant digits and the value is recorded. It is placed in a Kjeldahl
flask, washed with 50 mL of water, and added with 20 mL of sodium hydroxide solution (35→ 100). The flask is connected to a distillation device. 15 mL of boric acid solution (1 →25) and 3 drops of bromcresol green-methyl red test solution are put in a receiver and thenan appropriate amount of water is added in it. The bottom edge of the condenser isimmersed in this solution. It is distilled through steam until the volume of distillate liquidreaches 80~100 mL. The bottom edge of the condenser is lifted from the surface of thesolution and washed with a little water. The resulting mixed solution is titrated with 0.005
mol/L sulfuric acid. In this case, the end of titration is the time when the color of thesolution changes from green through light blue gray to light reddish violet gray. A blank test
is performed in the same manner and corrections are made.
0.005 mol/L sulfuric acid 1 mL = 1.221 mg C6H6N2O(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of nicotinamide isapplied mutatis mutandis.

(69) Calcium Lactate
A. Raw material for manufacturing(a) Compositional standards382

Provisional Translation from Japanese OriginalContent: When this product is determined after dried at 120 °C for 4 hours, it contains
98.0 % or more of calcium lactate (C6H10CaO6).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product dissolves in water and hardly dissolves in ethyl alcohol.
iii. This product is slightly weathered in air at room temperature and becomes anhydride
at 120 °C.
iv. The pH of a solution (1 → 20) of this product is 6~7.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of calcium salt and lactate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is added with 20 mL
of water and dissolved by warming. The resulting solution shall be colorless and clear.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed and added with 2 mL of
diluted acetic acid and approximately 35 mL of water, dissolved by warming, allowed
to cool, and added with water to make 50 mL. This is used as a sample solution. A
control solution is prepared using 2.0 mL of lead standard solution. When heavy metal
is tested using these solutions, the color of the sample solution shall not be darker thanthat of the control solution (20 mg/kg and less).
iii. Arsenic: 0.50 g (0.495~0.504 g) of this product is weighed and dissolved with 2 mLof water and 3 mL of hydrochloric acid to prepare a sample solution. When arsenic istested using the sample solution by the method using device A, the color of theabsorbing solution shall not be darker than the standard color (4 mg/kg or less).
iv. Volatile fatty acid: When 0.5 g (0.45~0.54 g) of this product is weighed, added with 1
mL of sulfuric acid, and warmed, the resulting solution shall not emit a fatty acid-likeodor.
Loss on drying: 25.0~30.0 % (2 g, 120 °C, 4 hours)
Assay: This product is dried at 120 °C for 4 hours, approximately 1.0 g of it is weighed tothe digits of 0.01 g, and the value is recorded. It is dissolved in 20 mL of dilutehydrochloric acid, transferred to a 100 mL volumetric flask, and added with water to thegraduation line to make 100 mL. 10 mL of the resulting solution is measured using a
volumetric pipette. It is added with 15 mL of potassium hydroxide (1 → 10), 3 mL ofpotassium cyanide solution (1 → 20), and 100 mL of water, allowed to stand forapproximately 1 minute, added with approximately 0.1 g of NN indicator, and titrated
with 0.05 mol/L ethylenediaminetetraacetic acid disodium solution within 30 seconds. In
this case, the end of titration is the time when the red color of the solution completely

383

Provisional Translation from Japanese Original

disappears and turns to blue. A blank test is performed in the same manner andcorrections are made.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL
= 10.91 mg C6H10CaO6(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium lactate areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium lactate
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of calcium
lactate and excipient.
Content: When this product is determined, it contains calcium lactate (C6H10CaO6)
corresponding to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1.0 g of the raw material for manufacturing of calcium lactate is weighed,added with 20 mL of water, shaken up well, and filtered. To the filtrate, the confirmation
test for the raw material for manufacturing of calcium lactate is applied mutatis mutandis.
Assay: The amount of this product containing approximately 1.0 g of calcium lactate(C6H10CaO6) is weighed and the value is recorded. It is added with 20 mL of dilutehydrochloric acid, shaken up well, and filtered. The residue on the filter paper is washedthree times with 20 mL of water each time. The mixture of the filtrate and the washings
are transferred to a 100 mL volumetric flask and added with water to the graduation line
to make 100 mL. 10 mL of the solution is measured using a volumetric pipette, addedwith 15 mL of potassium hydroxide solution (1 → 10), 3 mL of potassium cyanide
solution (1 → 20) and 100 mL of water, allowed to stand for approximately 1 minute, andadded with approximately 0.1 g of NN indicator, and hereinafter the assay for the raw
material for manufacturing of calcium lactate is applied mutatis mutandis.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL
= 10.91 mg C6H10CaO6

384

Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium lactate
is applied mutatis mutandis.

(70) P-Aminobenzoic Acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 2 hours, it contains
98.5 % or more of p-aminobenzoic acid (C7H7NO2).
Physical and chemical properties:
i. This product is white to pale yellow crystals or crystalline powder with no odor.
ii. This product is easy to dissolve in ethanol, easy to slightly dissolve in warm glycerin,slightly hard to dissolve in ether, and hard to dissolve in water or chloroform.
iii. This product is easy to dissolve in 1 mol/L sodium hydroxide test solution or sodium
carbonate test solution and slightly hard to dissolve in dilute hydrochloric acid.
iv. This product gradually turns to dark color by light or air.
Confirmation test:
i. 0.01 g (0.005~0.014 g) of this product is dissolved with 2 mL of dilute hydrochloric
acid and 3 mL of water. The resulting solution gives the qualitative reaction ofaromatic primary amine.
ii. When 0.5 g (0.45~0.54 g) of this product is dissolved with 1 mL of 1 mol/L sodiumhydroxide test solution and 1 mL of water and added with 0.5 mL of potassium iodide
test solution, 0.5 mL of dilute hydrochloric acid and 0.5 mL of sodium hypochlorite
test solution, brown precipitation is generated.
Purity test:
i. Melting point: The melting point of this product must be 186~189 °C
ii. Chloride: 0.5 g (0.45~0.54 g) of this product is weighed, added with 30 mL of waterand 10 mL of dilute nitric acid, dissolved by warming, and is added with water tomake 50 mL. This is used as a sample solution. By the chloride test method, a control
solution is prepared using 0.7 mL of 0.01 mol/L hydrochloric acid. When chloride istested, the opacity of the sample solution shall not be higher than that of the control
solution (0.05 % or less).
iii. Sulfate: 0.20 g (0.195~0.204 g) of this product is weighed, added with 30 mL ofwater, dissolved by warming, allowed to cool, and is added with 1 mL of dilutehydrochloric acid and water to make 50 mL. This is used as a sample solution. A

385

Provisional Translation from Japanese Originalcontrol solution is prepared using 1.0 mL of 0.005 mol/L sulfuric acid. When sulfate is
tested, the opacity of the sample solution shall not be higher than that of the control
solution (0.24 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
Loss on drying: 0.3 % or less (1 g, 105 °C, 2 hours)
Ignition residue: 0.2 % or less (1 g)Assay: This product is dried at 105 °C for 2 hours, approximately 0.3 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved with 5 mL of ethanol and
60 mL of water and titrated with 0.1 mol/L sodium hydroxide solution (indicator: 2 dropsof phenolphthalein test solution). A blank test is performed in the same manner andcorrections are made.
0.1 mol/L sodium hydroxide solution 1 mL = 13.71 mg C7H7NO2(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of p-aminobenzoic
acid are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of p-aminobenzoic
acid is applied mutatis mutandis.

(71) L-Valine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 3 hours, it contains
98.0 % or more of L-valine (C5H11NO2).
Physical and chemical properties:
i. This product is grayish white to grayish yellow brown crystals or crystalline powder
with a specific odor.
ii. The pH of a solution or aqueous suspension of this product (1 → 30) is 5.0~7.0.

386

Provisional Translation from Japanese OriginalConfirmation test:
i. 5 mL of a solution of this product in water (1 → 5,000) is added with 1 mL ofninhydrin test solution and heated for 3 minutes. The resulting solution is blue-violet
to purple.
ii. When 0.3 g (0.25~0.34 g) of this product is added with 10 mL of water, dissolved bywarming, added with 10 drops of dilute hydrochloric acid and 2 mL of sodium nitrite
test solution, it bubbles and generates colorless gas.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours, approximately 4 g it isweighed to the digits of 0.01 g, and the value is recorded. It is dissolved in 6 mol/Lhydrochloric acid test solution to make 50 mL, and filtered with a membrane filter(0.45 µm). The rotation of this solution shall be [α]20
D = +26.5 ~ +30.5°.

ii. Ammonium salt: The purity test iii. of the raw material for manufacturing ofaminoacetic acid is applied mutatis mutandis. In this case, “0.1 g” and “2 mL ofammonium standard solution” shall be replaced by “0.02 g” and “3 mL of ammonium
standard solution,” respectively (0.15 % or less).
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Arsenic: The purity test of the raw material for manufacturing of L-isoleucine isapplied mutatis mutandis.
Loss on drying: 1.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 1.0 % or less (1 g)Assay: This product is dried at 105 °C for 3 hours, approximately 0.25 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved in 3 mL of formic acid,added with 50 mL of glacial acetic acid for nonaqueous titration, and titrated with 0.1
mol/L perchloric acid (indicator: 1 mL of α-naphtholbenzein test solution). In this case,the end of titration is the time when the color of the solution changes from orange-yellow
through yellowish green to green. A blank test is performed in the same way andcorrections are made.
0.1 mol/L perchloric acid 1 mL = 11.72 mg C5H11NO2(b) Standard of storage method
It shall be stored in a lightproof capped container.

387

Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-valine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-valine isapplied mutatis mutandis.

(72) Calcium D-Pantothenate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried at 105 °C for 4 hours and determined, it contains
5.7~6.0 % of nitrogen (N) and 8.2~8.6 % of calcium (Ca).
Physical and chemical properties:
i. This product is white powder with no odor and with a bitter taste.
ii. This product is easy to dissolve in water, slightly hard to dissolve in glycerin,
extremely hard to dissolve in ethanol, and hardly dissolves in ether and chloroform.
iii. The pH of a solution of this product in water (1 → 20) is 7.0~9.0.
iv. A solution of this product in water (1 → 20) has an optical rotation.
v. This product is hygroscopic.
Confirmation test:
i. 0.05 g (0.045~0.054 g) of this product is dissolved with 5 mL of 1 mol/L sodiumhydroxide test solution and filtered. When the filtrate is added with a drop of copper
sulfate test solution, the resulting solution is dark blue.
ii. 0.05 g (0.045~0.054 g) of this product is dissolved with 5 mL of 1 mol/L sodiumhydroxide test solution, boiled for 1 minute, allowed to cool, added with hydrochloric
acid (1 → 10) to make the solution pH 3 to 4, and added with 2 drops of ferric chloride
test solution. The resulting solution is yellow.
iii. A solution of this product in water (1 → 10) gives the qualitative reactions of calcium
salt.
Purity test:i. Specific rotation: This product is dried at 105 °C for 4 hours, approximately 1 g of it isweighed to the digits of 0.1 g, and the value is recorded. It is dissolved in water tomake 20 mL. The rotation of this solution shall be [α]20= +25.0 ~ +28.5°.
D388

Provisional Translation from Japanese Originalii. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and clear.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 1 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Alkaloid: 0.05 g (0.045~0.054 g) of this product is dissolved with 5 mL of water, 1
mL of dilute hydrochloric acid and 2 drops of Mayer's test solution, and allowed tostand for 1 minute. The resulting solution shall not produce opacity.
Loss on drying: 5.0 % or less (1 g, 105 °C, 4 hours)
Assay:
i. Nitrogen: This product is dried at 105 °C for 4 hours, approximately 0.05 g of it isweighed to the digits of 0.01 mg, and the value is recorded. The test is performed by
the nitrogen determination method (Semimicro-Kjeldahl Method).
ii. Calcium: This product is dried at 105 °C for 4 hours, approximately 0.4 g of it isweighed to the digits of 0.001 g, and the value is recorded. It is added with 30 mL ofwater, dissolved by warming, allowed to cool, added with 25 mL of 0.05 mol/L
ethylenediaminetetraacetic acid disodium solution using a volumetric pipette, addedwith 10 mL of pH 10.7 ammonia-ammonium chloride buffer and 0.04 g (0.035~0.044g) of eriochrome black T-sodium chloride indicator, and an excess amount of
ethylenediaminetetraacetic acid disodium is titrated with 0.05 mol/L magnesium
chloride solution. In this case, the end of titration is the time when the color of thesolution changes from blue-violet to reddish violet. A blank test is performed in thesame way.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 2.004 mg Ca(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium Dpantothenate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium Dpantothenate is applied mutatis mutandis.

389

Provisional Translation from Japanese Original(73) Calcium DL-Pantothenate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried at 105 °C for 4 hours and determined, it contains
5.7~6.0 % of nitrogen (N) and 8.2~8.6 % of calcium (Ca).
Physical and chemical properties:
i. This product is white powder with no odor and with a bitter taste.
ii. This product is easy to dissolve in water, slightly hard to dissolve in glycerin,
extremely hard to dissolve in ethanol, and hardly dissolves in ether and chloroform.
iii. The pH of a solution of this product in water (1 → 20) is 7.0~9.0.
iv. A solution of this product in water (1 → 20) does not have an optical rotation.
v. This product is hygroscopic.
Confirmation test: The confirmation test of the raw material for manufacturing of calcium
D-pantothenate is applied mutatis mutandis.
Purity test:
i. Clarity and color of solution: The purity test ii, of the raw material for manufacturingof calcium D-pantothenate is applied mutatis mutandis.
ii. Heavy metal: The purity test iii, of the raw material for manufacturing of calcium Dpantothenate is applied mutatis mutandis (20 mg/kg or less).
Loss on drying: 5.0 % or less (1 g, 105 °C, 4 hours)
Assay: The assay of the raw material for manufacturing of calcium D-pantothenate isapplied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium DLpantothenate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium DLpantothenate is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of calcium
DL-pantothenate and excipient.

390

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains nitrogen (N) and calcium (Ca)corresponding to calcium DL-pantothenate (C18H32CaN2O5) corresponding to 90~110 %of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.05 g of
the raw material for manufacturing of calcium DL-pantothenate is weighed and theconfirmation test i. for the raw material for manufacturing of calcium D-pantothenate
is applied mutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 0.05 g of
the raw material for manufacturing of calcium DL-pantothenate is weighed, addedwith 5 mL of sodium hydroxide test solution, shaken up well, and filtered. To thefiltrate, the confirmation test ii. for the raw material for manufacturing of calcium Dpantothenate is applied mutatis mutandis.
iii. According to the amount of this product on the label, the amount containing 1.0 g of
the raw material for manufacturing of calcium DL-pantothenate is weighed, addedwith water to make 10 mL, shaken up well, and filtered. To the filtrate, theconfirmation test iii. for the raw material for manufacturing of calcium D-pantothenate
is applied mutatis mutandis.
Assay:
i. Nitrogen: The amount of this product containing approximately 0.05 g of calcium DLpantothenate (C18H32CaN2O5) is weighed to three significant digits and the value isrecorded. The determination of nitrogen for the raw material for manufacturing of
calcium D-pantothenate is applied mutatis mutandis.
ii. Calcium: The amount of this product containing approximately 0.4 g of calcium DLpantothenate (C18H32CaN2O5) is weighed to three significant digits and the value isrecorded. It is added with 10 mL of water, warmed, shaken up well, and filtered. The
residue on the filter paper is washed twice with 10 mL of water each time. To the
mixture of the filtrate and the washings, the determination of calcium of the raw
material for manufacturing of calcium D-pantothenate is applied mutatis mutandis.
0.05 mol/L ethylenediaminetetraacetic acid disodium = 2.004 mg Ca(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium DLpantothenate is applied mutatis mutandis.

391

Provisional Translation from Japanese Original(74) d-Biotin
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined after dried at 105 °C for 4 hours, it contains
98.0 % or more of d-biotin (C10H16N2O3S).
Physical and chemical properties:
i. This product is white to pale yellow powder with no odor.
ii. This product is hard to dissolve in water, extremely hard to dissolve in ethanol, andhardly dissolves in acetone, ether, and chloroform.
iii. Melting point: 228~232 °C (degradation)
Confirmation test:
i. 5 mg (4.5~5.4 mg) of this product is added with 5 mL of dilute sodium hydroxide test
solution and then added with 3 drops of potassium permanganate test solution. The
resulting solution changes from reddish violet through blue to verdigris color.
ii. 5 mL of a solution of this product in ethanol (1 → 10,000) is added with 1 mL of asolution of sulfuric acid in ethanol (2 → 100), then added with 1 mL of pdimethylaminocinnamaldehyde, and is allowed to stand for an hour. The resulting
solution is orange-red.
Purity test:i. Specific rotation: 0.5 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved in dilute sodium hydroxide test solution to make 50 mL.
The rotation of this solution measured at a layer length of 100 mm shall be [α]20D =+90 ~ +94°.

ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Loss on drying: 0.5 % or less (0.5 g, 105 °C, 4 hours)
Ignition residue: 0.1 % or less (0.5 g)
Assay: This product is dried at 105 °C for 4 hours, approximately 0.4 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is added with 10 mL of392

Provisional Translation from Japanese Original

dimethylformamide and heated on a water bath to dissolve. It is cooled down to room
temperature, added with 30 mL of absolute ethanol, shaken up well, and titrated with 0.1
mol/L of sodium methoxide solution (indicator: 0.4 mL of thymol blue test solution).
This time, the end of titration is the time when the color of the solution changes fromyellow to verdigris color. A blank test is performed in the same manner and corrections
are made.
0.1 mol/L of sodium methoxide solution 1 mL = 24.43 mg C10H16N2O3S(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof d-biotin and excipient.
Content: When this product is determined, it contains d-biotin (C10H16N2O3S)
corresponding to 90~120 % of the amount on the label.
Conformation test: This product is powdered. According to the amount on the label, theamount of the product containing 4 mg of the raw material for manufacturing of d-biotin
is weighed, added with 1 mL of dimethylformamide, shaken up, added with 20 mL ofethanol, warmed for 10 minutes, and filtered. 5 mL of the filtrate is added with 1 mL ofabsolute ethanol solution (2 → 100), then added with 1 mL of a solution of pdimethylaminocinnamaldehyde in absolute ethanol (2 → 1,000), and allowed to stand foran hour. The resulting solution is orange-red.
Assay: This product is powdered to prepare a sample. Method No. 2 is used for a
preparation diluted with dextrin or those added with organic acids including sorbic acidas a preservative, and Method No. 1 is used for others.
Method No. 1: The amount of this product containing approximately 3 mg of d-biotin(C10H16N2O3S) is weighed to three significant digits and the value is recorded. It isplaced in a flask and added with 30 mL of absolute ethanol. The flask is attached to a
reflux condenser and heated in a bath for 45 minutes. Then, it is cooled down to room
temperature and transferred to a 50 mL volumetric flask. The flask is washed with 10
mL of absolute ethanol and the washings are transferred to the volumetric flask.
Absolute ethanol is added to the graduation line to make 50 mL and the resulting
solution is filtered. 20 mL of the first filtrate is removed and the next filtrate is used asa sample solution. Separately, approximately 6 mg of d-biotin for assay is weighed tothe digits of 0.01 mg and the value is recorded. It is dissolved with absolute ethanol,transferred to a 100 mL volumetric flask and added with more absolute ethanol to the393

Provisional Translation from Japanese Original

gradation line to make 100 mL. This is used as a standard solution. 2 mL each of thesample and standard solutions is measured using a volumetric pipette and placed in a
20 mL volumetric flask, and added with 2 mL of a solution of sulfuric acid in absoluteethanol (2 → 100) and 2 mL of a solution of p-dimethylaminocinnamaldehyde inabsolute ethanol (2 → 1,000) using a volumetric pipette. It is added with moreabsolute ethanol to the graduation line to make 20 mL, shaken up, and allowed to standfor 1 hour. Separately, 2 mL of absolute ethanol is measured, and subjected to thesame procedure to prepare a control solution. The absorbances of AT and AS at the
maximum wavelength around 530 nm are measured.
Amount of d-biotin (C10H16N2O3S) (mg)
= Amount of d-biotin for assay (mg) ×

AT 1
×
AS 2

Method No. 2: The amount of this product containing approximately 1 mg of d-biotin(C10H16N2O3S) is weighed to three significant digits and the value is recorded. It is
added with 30 mL of ammonia solution (8 → 40), added with 150 mL of sodium
thiosulfate solution (2 → 1,000) within 30 seconds, allowed to stand for 30 minuteswith occasional shaking. Then it is transferred to a 1 L volumetric flask, added withwater to the graduation line to make 1 L, and filtered. 50 mL of the filtrate is measured
using a volumetric pipette, adjusted with 0.1 mol/L hydrochloric acid test solution to
make the solution pH 9.0, transferred to a 100 mL volumetric flask, and added withwater to make 100 mL. This is used as a sample solution. Separately, approximately 10
mg of d-biotin for assay is weighed to the digits of 0.1 mg and the value is recorded. It
is dissolved with 1 mL of ammonia solution (8 → 40), transferred to a 100 mL
volumetric flask, and added with water to the graduation line to make 100 mL. This isused as a standard stock solution. 1 mL of the standard stock solution is measured
using a volumetric pipette, transferred to a 200 mL volumetric flask, and added withwater to the graduation line to make 200 mL. This is used as a standard solution(prepared at time of use). The sample and standard solutions are tested by the
microbioassay under the following conditions.
Culture media:
i. Agar medium for storing test strain
Yeast extract: 20 g
Glucose: 10 g
Peptone: 5 gPolysorbate 80: 0.1 g
Potassium dihydrogenphosphate: 2 g

394

Provisional Translation from Japanese Original

Agar: 10 g
The materials mentioned above are weighed, added with water to make 1 L, andsterilized. The pH shall be 6.7.
ii. Medium for enrichment of test strain
Yeast extract: 20 g
Glucose: 10 g
Peptone, casein: 5 gPolysorbate 80: 0.1 g
Potassium dihydrogenphosphate: 2 g
The materials mentioned above are weighed, added with water to make 1 L, andsterilized. The pH shall be 6.7.
iii. Basal medium for determination
Glucose: 40 g
Sodium acetate: 20 g
Casamino acid: 14 g
L-cystine: 400 mg
DL-tryptophan: 200 mg
Uracil: 20 mg
Guanine hydrochloride: 20 mg
Adenine sulfate: 20 mg
Thiamine hydrochloride: 0.8 mg
Pyridoxine hydrochloride: 0.8 mg
Nicotinic acid: 0.4 mg
Calcium pantothenate: 0.4 mg
Riboflavin: 0.4 mg
p-aminobenzoate: 0.2 mg
Dibasic potassium phosphate: 1 g
Potassium dihydrogenphosphate: 1 g
Magnesium sulfate: 400 mg
Ferrous sulfate: 20 mg
Manganese sulfate: 20 mg
The materials mentioned above are weighed, added with water to make 500 mL andsterilized and the pH shall be 6.7 to 6.9. Separately, an agar solution (3 → 100) is
prepared and sterilized. These solutions are mixed in equal amounts in heating, andstored in a water bath at 45 °C.
Preparation of test strain and test strain culture:395

Provisional Translation from Japanese Original

Lactobacillus plantarum ATCC 8014 is used as a test strain. 5 mL of agar medium forstoring test strain is added in a test tube to prepare a slant medium. The test strain in the test
tube is subcultured at about monthly intervals (37 °C, 18~24 hours) and stored at 4~8 °C.
This stored test strain is added with 5 mL of sterile physiological saline to make thebacterial cells uniformly suspended. 0.5~1.0 mL of it is inoculated into 30 mL of a medium
for enrichment of test strain and cultivated at 37 °C for 18~20 hours. After the cultivation, it
is centrifuged to make the bacterial cells precipitated and the supernatant fluid is removed.
The precipitated bacterial cells are added with 30 mL of sterile physiological saline, shaken
up, and centrifuged again. This procedure is performed once again and then the bacterial
cells are uniformly suspended in 5 mL of sterile physiological saline. This is used as a test
strain culture.
Procedures:
80 mL of the basal medium for determination is added with 4 mL of the test strain culture
to mix them. The mixture is poured into a sterile plate (20 × 20 cm) preheated at 50 °C toprepare a uniform plate medium. Six sets of discs, provided that a set has 8 discs with adiameter of 6 mm, are prepared and each of them is called SL, SM, SH, UL, UM and UH. 5, 10
and 20 µL of the standard solution are spotted on the discs SL, SM and SH, respectively, and5, 10 and 20 µL of the sample solution are spotted on the discs UL, UM and UH, respectively.
One each of the discs of each set is placed on the plate medium at intervals of 3 cm or more.
A total of 8 plate media are prepared in this way, and cultured at 37 °C for 16~20 hours.
Calculation method:
The diameters of the growth obtained from the discs of the plate culture, SL, SM, SH, UL,
UM and UH are measured to 0.1 mm. Each of them is called y1, y2, y3, y4, y5 and y6. y1, y2, y3,y4, y5 and y6 of each plate medium are summed, and each of them is called Y1, Y2, Y3, Y4,
Y5 and Y6.
Amount of d-biotin (C10 H16 N2O3S) (mg)
= Amount of reference standard of d-biotin (mg) × antilogM ×
M = - 0.4013

YA
YB

YA = Y1 + Y2 + Y3 − Y4 − Y5 − Y6
YB = − Y1 + Y3 − Y4 + Y6

396

1
10

Provisional Translation from Japanese Original

Where, VA, VB, VC and S2 are calculated using the following equation,
VC
S2

VA

VB

S

S2

2 ,

and

, each of them shall be 4.05 or less. If the value is exceeded, the experiment shallbe repeated under improved experimental conditions until the value is obtained.
VA =(Y1 - 2Y2 + Y3 + Y4 - 2Y5 + Y6 )2
96

VB =

(- Y + Y3 + Y - Y6 )2
32

VC =

(Y1 - 2Y2 + Y3 - Y4 + 2Y5 - Y6 )2
96

S2 =

ST - SD42

ST = Σy2 -( Σy )
422(Σy2: y1, y2, y3, y4, y5 and y6 each of each set is squared, and theobtained values are summed.)SD =( YA )48

2

2+ ( Y32B ) + VA + VB + VC(b) Standard of storage method
It shall be stored in a lightproof capped container.

(75) Vitamin A Powder
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it is 200,000 vitamin A international unit or more
per 1 gram and contains vitamin A (retinol) corresponding to 90~130 % of the unit on thelabel.
Physical and chemical properties:
i. This product is pale yellow to red-brown powder with no odor or a specific odor.
ii. Degradation of this product is promoted by air or light.
Confirmation test: 2 mL of isopropanol solution obtained in the section of Assay ismeasured and isopropanol is distilled away under reduced pressure. The residue isdissolved with 1 mL of chloroform, and added with 3 mL of antimony trichloride test
solution. The resulting solution is blue within 30 seconds but the color disappears within
30 seconds.

397

Provisional Translation from Japanese Original

Purity test:
i. Rancidity: This product shall not emit an unpleasant odor.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed. The residue obtained
according to the ignition residue test method is added with 1 mL of hydrochloric acidand 0.2 mL of nitric acid, evaporated to dryness on a water bath, added with 1 mL of
dilute hydrochloric acid and 15 mL of water, heated to dissolve, allowed to cool, addedwith a drop of phenolphthalein test solution, added with ammonia test solution by
dropping until the solution becomes slightly red, added with 2 mL of diluted acetic
acid, filtered as appropriate, and washed with 10 mL of water. The filtrate and thewashings are put in a Nessler tube and added with water to make 50 mL. This is used
as a sample solution. The sample solution is added with 2 drops of sodium sulfide test
solution and allowed to stand for 5 minutes. The color of this solution shall not be
darker than that of a solution prepared in the procedure in which 5.0 mL of leadstandard solution is added with 2 mL of diluted acetic acid and water to make 50 mL,
added with 2 drops of sodium sulfide test solution and allowed to stand for 5 minutes(50 mg/kg or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed into a decomposition flask,
added with 20 mL of nitric acid, weakly heated until the content becomes fluid. It isallowed to cool, added with 5 mL of sulfuric acid, and heated until white smoke
emerges. When the solution is still brown, it is allowed to cool and added with 5 mL ofnitric acid and heated. This procedure is repeated until the solution is colorless to paleyellow. It is allowed to cool, added with 15 mL of saturated ammonium oxalate
solution and heated until white smoke emerges. It is allowed to cool and added withwater to make 25 mL. 5 mL of this solution is measured and used as a sample solution.
When arsenic is tested using the sample solution by the method using device A, thecolor of the absorbing solution shall not be darker than the standard color. In this case,the standard color is prepared by way in which 4.0 mL of arsenic standard solution ismeasured into a decomposition flask, added with 20 mL of nitric acid, and hereinafter
subjected to the same procedures for the sample solution (4 mg/kg or less).
Loss on drying: 5.0 % or less (1 g, reduced pressure, silica gel, 4 hours)
Assay: Approximately 0.1 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is placed in a flask, added with 2 mL of water, warmed for a while withshaking, and tested by Method No. 2 of the vitamin A assay.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of vitamin A oil, retinol acetate,or retinol palmitate is powdered or coated and granulated using appropriate base materials.

398

Provisional Translation from Japanese Original(c) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of vitamin A powder
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofvitamin A powder and excipients.
Content: When this product is determined, it contains vitamin A (retinol) corresponding to
90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
Assay: The assay of the raw material for manufacturing of vitamin A powder is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.

(76) Vitamin A Oil
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it is 30,000 vitamin A international unit or more
per 1 gram and contains vitamin A (retinol) corresponding to 90~120 % of the unit on thelabel.
Physical and chemical properties:
i. This product is yellow to yellow-brown, clear or slightly opacified oil liquid with noodor or a slight specific odor.
ii. Degradation of this product is promoted by air or light.
Confirmation test: This product is dissolved in chloroform to prepare a solution containing
30 vitamin A international unit per 1 mL according to the unit on the label. When 1 mL

399

Provisional Translation from Japanese Originalof this solution is measured, added with 3 mL of antimony trichloride test solution, theresulting solution is blue within 30 seconds but the color disappears within 30 seconds.
Purity test:
i. Ultraviolet absorption spectrum: This product shall be fit for the conditions which canbe measured by Method No. 1 of the vitamin A assay. Or, the f value shall be 0.85 ormore when it is measured by Method No. 2 of the vitamin A assay.
ii. Acid: 1.2 g (1.15~1.24 g) of this product is weighed and added with 30 mL of a mixed
solution of neutralized ethanol and ether (1:1), gently boiled for 10 minutes with a
reflux condenser, allowed to cool, added with 5 drops of phenolphthalein test solution
and 0.60 mL of 0.1 mol/L sodium hydroxide solution. The resulting solution shall be
red.
iii. Rancid: When this product is warmed, it shall not emit an unpleasant rancid odor.
Assay: Test is performed by Vitamin A quantification method.
(b) Standard of manufacturing method
It is manufactured from fatty oil from fresh livers or pyloric appendages of aquatic
animals, or manufactured by the procedure in which the fatty oil, its concentrate, or fatty
acid ester of vitamin A is added with cod liver oil or vegetable oil.
(c) Standard of storage method
This product shall be stored in a lightproof airtight container. The container shall bealmost full of this product or the air in the container shall be replaced by N2.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of vitamin A oil areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin A oil isapplied mutatis mutandis.
C. Preparation (Part 2 Liquid)(a) Compositional standards
This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of vitamin A oil and hydrogenated oils, higher saturated fatty acids, fatty
acids, vegetable oil or animal fats.
Content: When this product is determined, it contains vitamin A (retinol) corresponding to
90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.

400

Provisional Translation from Japanese Original

Assay: The assay of the raw material for manufacturing of vitamin A powder is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin A oil isapplied mutatis mutandis.
D. Preparation (Part 3 Powder)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofvitamin A oil and fillers.
Content: When this product is determined, it contains vitamin A (retinol) corresponding to
90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
Assay: The assay of the raw material for manufacturing of vitamin A powder is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.

(77) Vitamin D Powder
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it is 100,000 vitamin D international unit or more
per 1 gram and contains cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
corresponding to 90~130 % of the unit on the label.
Physical and chemical properties:
i. This product is grayish white to red-brown powder with little odor.
ii. Degradation of this product is promoted by air or light.
Confirmation test:
i. When 10 µL of the sample solution for thin-layer chromatography obtained by thevitamin D assay is measured, spotted in parallel with solutions containing vitamin D
and previtamin D on the same thin-layer plate, developed, and irradiated withultraviolet light (dominant wavelength: 254 nm), the spots of vitamin D andprevitamin D are dark purple and their Rf values are equal.

401

Provisional Translation from Japanese Originalii. Peak retention times of vitamin D with low retention time and isopiro vitamin D andstigmasterol acetate, which are with high retention times, are determined from thechromatograms from gas chromatography of the sample and standard solutions
obtained by the vitamin D assay. Their relative retentions to that of stigmasterol
acetate are equal.
Purity test:
i. Rancid: This product shall not emit an unpleasant odor.
ii. Heavy metal: The purity test ii. for the raw material for manufacturing vitamin A
powder is applied mutatis mutandis (50 mg/kg or less).
iii. Arsenic: The purity test iii. for the raw material for manufacturing vitamin A powder
is applied mutatis mutandis (4 mg/kg or less).
Loss on drying: 7.0 % or less (1 g, reduced pressure, silica gel, 4 hours)
Assay: Test is performed by Vitamin D quantification method.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of cholecalciferol, the raw
material for manufacturing of ergocalciferol or solutions of them in edible vegetable oil, or
vitamin D3 oil is powdered, or coated and granulated using appropriate base materials.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of vitamin D powder
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofvitamin D powder and fillers.
Content: When this product is determined, it contains cholecalciferol (C27H44O) or
ergocalciferol (C28H44O) corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.
Assay: Test is performed by Vitamin D quantification method.
(b) Standard of storage method402

Provisional Translation from Japanese Original

The standard of storage method of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.

(78) Vitamin D3 Oil
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it is 200,000 vitamin D international unit or more
per 1 gram and contains cholecalciferol (C27H44O) corresponding to 90~130 % of the unit
on the label.
Physical and chemical properties:
i. This product is pale yellow to red-brown, clear or slightly opacified viscous oil liquidor waxy blocks with no odor or a slight specific odor.
ii. Degradation of this product is promoted by air or light.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.
Purity test:i. Acid: The purity test ii. for the raw material for manufacturing vitamin A oil is appliedmutatis mutandis.
ii. Rancidity: When this product is warmed, it shall not emit an unpleasant rancid odor.
Assay: Test is performed by Vitamin D quantification method.
(b) Standard of manufacturing method
For manufacturing, vitamin D3 is added with edible vegetable oil.
(c) Standard of storage method
This product shall be stored in a lightproof airtight container. The container shall bealmost full of this product or the air in the container shall be replaced by N2.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of vitamin D3 oil areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin D3 oil isapplied mutatis mutandis.
C. Preparation (Part 2 Liquid)(a) Compositional standards403

Provisional Translation from Japanese Original

This product is oil liquid or water-soluble liquid of the mixture of the raw material for
manufacturing of vitamin D3 oil and hydrogenated oils, higher saturated fatty acids, fatty
acids, vegetable oil or animal fats.
Content: When this product is determined, it contains cholecalciferol (C27H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.
Assay: Test is performed by Vitamin D quantification method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin D3 oil isapplied mutatis mutandis.
D. Preparation (Part 3 Powder)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofvitamin D3 oil and excipients.
Content: When this product is determined, it contains cholecalciferol (C27H44O)
corresponding to 90~130 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.
Assay: Test is performed by Vitamin D quantification method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin D
powder is applied mutatis mutandis.

(79) Vitamin E Powder
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 100 mg or more of dl-α-tocopheryl
acetate per 1 gram and contains dl-α-tocopheryl acetate (C31H52O3) corresponding to95~120 % of the unit on the label.
Physical and chemical properties:
i. This product is yellowish white to brown powder with little odor.
ii. Degradation of this product is promoted by air or light.

404

Provisional Translation from Japanese OriginalConfirmation test: 10 mL of the sample solution obtained by the assay method is measured,
added with 2 mL of nitric acid and heated at 75 °C for 15 minutes. The resulting solution
is red to orange.
Purity test:
i. Rancid: This product shall not emit an unpleasant odor.
ii. Heavy metal:
Method No. 1: The purity test ii. for the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis. However, the residue on ignition of this productis added with 1 mL of hydrochloric acid and 0.2 mL of nitric acid, evaporated todryness on a water bath. Then it is added with 1 mL of dilute hydrochloric acid and 15
mL of water, dissolved by heating. If there are insoluble matters, Method No. 2 shallbe applied (50 mg/kg or less).
Method No. 2: 1.0 g (0.95~1.04 g) of this product is weighed into a platinum pot witha loose lid and weakly heated to be carbonized. It is allowed to cool, added with 2 mLof nitric acid and 5 drops of sulfuric acid, carefully heated until white smoke emerges,
and ignited at 500~600 °C to be converted into ashes. After cooling, the residue ismoisturized with water, added with 6 mL of hydrofluoric acid and 3 drops of sulfuric
acid, evaporated to dryness, and ignited for 5 minutes. It is allowed to cool, addedwith 2 mL of hydrochloric acid, evaporated to dryness on a water bath, moisturizedwith 3 drops of hydrochloric acid, added with 10 mL of boiling water, and heated for 2
minutes to dissolve. It is allowed to cool, added with a drop of phenolphthalein test
solution, added with ammonia test solution by dropping until the solution becomesslightly red, added with 2 mL of diluted acetic acid, filtered as appropriate, andwashed with 10 mL of water. The filtrate and the washings are transferred to a Nessler
tube and added with water to make 50 mL. This is used as a sample solution. When
this sample solution is added with 2 drops of sodium sulfide test solution, and allowed
to stand for 5 minutes, the color of the solution shall not be darker than that of thesolution prepared by the following procedure: 2 mL of nitric acid, 8 drops of sulfuric
acid, 2 mL of hydrochloric acid, 6 mL of hydrofluoric acid, and 2 mL of diluted acetic
acid are evaporated on a water bath, and then evaporated to dryness on a sand bath.
Then, the residue is moisturized with 3 drops of hydrochloric acid, added with 10 mLof boiling water, and hereinafter subjected to the preparation method of the sample
solution, added with 5.0 mL of lead standard solution and water to make 50 mL, addedwith 2 drops of sodium sulfide test solution and allowed to stand for 5 minutes (50
mg/kg or less).

405

Provisional Translation from Japanese Originaliii. Arsenic: The purity test iii. for the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis (4 mg/kg or less).
Loss on drying: 7.0 % or less (1 g, reduced pressure, silica gel, 4 hours)
Assay: The amount of this product containing approximately 0.02 g of dl-α-tocopheryl
acetate is weighed to three significant digits and the value is recorded. It is saponified by
the vitamin A assay, extracted, and dehydrated. The ether extract is evaporated withshaking in water at 45 °C using an aspirator. The residue is dissolved in ethanol within 30
seconds, transferred to a 100 mL volumetric flask, and added with ethanol to thegraduation line to make 100 mL. 5 mL of this solution is measured using a volumetricpipette, transferred to a 50 mL volumetric flask, and added with ethanol to the graduationline to make 50 mL. This is used as a sample solution. Separately, approximately 0.02 gof dl-α-tocopheryl acetate for assay is weighed to the digits of 0.1 mg, the value isrecorded, and it is subjected to the preparation procedure of the sample solution toprepare a standard solution. 10 mL each of the sample and standard solutions is measured
using a volumetric pipette, transferred to a 25 mL volumetric flask, added with 1 mL of
dilute ferric chloride test solution and 1 mL of α-α'-dipyridyl test solution, and added withethanol to the graduation line to make 25 mL. Separately, 10 mL of ethanol is measured
using a volumetric pipette, and subjected to the same procedures. The resulting solution isused as a control solution, and the absorbance of AT and AS at a wavelength 520 nm is
measured.
Amount of dl-α-tocopheryl acetate (C31H52O3) (mg)
= Amount of dl-α-tocopheryl acetate for assay (mg) ×ATAS(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of dl-α-tocopheryl acetate ispowdered or coated and granulated using appropriate base materials.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of vitamin E powder
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.

406

Provisional Translation from Japanese Original

C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofvitamin E powder and fillers.
Content: When this product is determined, it contains dl-α-tocopheryl acetate (C31H52O3)
corresponding to 90~120 % of the amount on the label.
Confirmation test: The confirmation test of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.
Assay: The assay of the raw material for manufacturing of vitamin E powder is appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of vitamin E
powder is applied mutatis mutandis.

(80) 25-Hydroxycholecalciferol
A. Raw material for manufacturing(a) Compositional standards
Content: When quantified, this product contains 25-hydroxycholecalciferol (C27H44O2·H2O) at
no less than 94.0 %.
Physical and chemical properties:
i. This product is white crystal.
ii. This product changes in air or light.
Confirmation test:
i. When 20 μL of a sample solution prepared by a quantification method and a standard
solution is tested by liquid chromatography under the operating condition of thequantification method, the retention time pertaining to the peak of 25hydroxycholecalciferol (C27H44O2·H2O) obtained from the sample solution is the same asthat obtained from the standard solution.
ii. When this product and a 25-hydroxycholecalciferol reference standard are measured byinfrared absorption spectroscopy using the potassium bromide pellet method and the spectra
are compared, absorptions are observed at the same wavenumbers and the relative intensityof the absorptions is the same.
Purity test:
i. Erythrosine Weigh approximately 1.0 g of this product to the digit of 0.01 g, record thevalue, add methanol to dissolve, place in a 10 mL volumetric flask, and further add

407

Provisional Translation from Japanese Original

methanol to the marked line (i.e. volume up to 10 mL) to prepare a sample solution. For thissolution, measure the absorbance, A1, at wavelength 530 nm using methanol as thereference. Next, for 10 mL of methanol, perform the same procedure as that for the sample
solution, and measure the absorbance, A0. When the content of ionized erythrosine iscalculated by the equation below, the amount shall be no more than 5 μg/g.
Erythrosine content (μg/g) =(A1 - A0) / ε×833. 9×106wTwT : Weight of this product (g)ε : Molar absorption coefficient of erythrosine at 530 nm 110,000 (L/mol·cm)
ii. Analogous substances Measure 5 mL of the standard stock solution prepared by aquantification method using a volumetric pipette, place in a 100-mL volumetric flask, andadd methanol to the marked line (i.e. volume up to 100 mL) to prepare the standard stock
solution for purity test. Filter the standard stock solution for a purity test with a membranefilter (0.45 μm) to prepare a standard solution for the purity test. For 20 μL of a sample
stock solution prepared by the quantification method and the standard solution for a purity
test, perform testing by liquid chromatography under the conditions below. From thechromatograms obtained, identify the peak of each sterol that appears in the chromatogramof sample stock solution using a reference chromatogram of this product, and calculate thepeak area, AT. When the content of analogous substances of 25-hydroxycholecalciferol(which refer to sterols originating from manufacturing of 25-hydroxycholecalciferol otherthan 25-hydroxycholecalciferol; the same shall apply to ii. hereinafter) is calculated fromthis value and the peak area, AS, of 25-hydroxycholecalciferol calculated from thechromatogram of the standard solution for the purity test using the equation below, theamount shall be no more than 1 % for each analogous substance.
Reference chromatogram of this product408

Provisional Translation from Japanese OriginalContent of each analogous substance (%) = p ×

ws
wT

×

AT
AS

×F×2

p: Purity of 25-hydroxycholecalciferol reference standard
wT : Weight of this product (mg)ws : Weight of 25-hydroxycholecalciferol reference standard (mg)
AT: Peak area of sterol in the sample solution
AS : Peak area of 25-hydroxycholecalciferol in the standard solution
F: Absorption coefficient of each analogous substance specified in the table below
Table
Analogous substance

Absorption coefficient

25-Hydroxyisotachysterol

0.830025-Hydroxyisovitamin D3

0.830025-Hydroxycholecalciferol

1.000025-Hydroxytachysterol

0.910925-Hydroxy-5,6-trans-vitamin D3

0.898625-Hydroxyprovitamin D3

1.7677Pre-25-hydroxycholecalciferol

2.3863

Unknown sterol which shows a similar spectrum with

1.000

25-hydroxycholecalciferol

409

Provisional Translation from Japanese Original

Operating condition
Detector: Photodiode array detector (measuring wavelength: 230~330 nm (measure atwavelength 270 nm for quantification))
Column: Fill a stainless steel tube of 4.6 mm in inner diameter and 250 mm in length withoctadecylsilyl silica gel for liquid chromatography of no more than 5 μm in particle
diameter.
Column temperature: A constant temperature around 28 °C
Mobile phase: Methanol-acetonitrile-water mixture solution (55:22:23)
Flow rate: Approximately 1.0 mL per minute
Measurement time: 120 minutes
Column selection: Heat 10 mL of the standard stock solution for purity test at 50~55°C for
2 hours to generate pre-25-hydroxycholecalciferol and filter with a membrane filter (0.45μm). When the operation above is applied to 20 μL of this filtrate, the column to be usedshall elute in the order of 25-hydroxycholecalciferol and then pre-25hydroxycholecalciferol and the degree of separation shall be no less than 4.0.
iii. Lead Weigh 0.5 g (0.45~0.54 g) of this product and when a test for lead is performed by
the limit test for lead (atomic absorption spectrometry No.1), the amount shall be no morethan 20 μg/g.
iv. Aluminum Weigh 0.2 g (0.15~0.24 g) of this product, place in a platinum or quartz
crucible, wet by adding a small amount of sulfuric acid, gradually heat to incinerate at as
low a temperature as possible, allow to cool, further add 1 mL of sulfuric acid, gradually
heat, and ignite until incinerated at 450~550 °C. Dissolve the residue by adding a small
amount of nitric acid (1 → 150), place in a 50 mL, and add water to the marked line (i.e.
volume up to 50 mL) to prepare the sample solution. Separately, measure 1 mL of analuminum standard solution using a volumetric pipette, place in a 100 mL volumetricflask, and add water to the marked line (i.e. volume up to 100 mL). Measure 1 mL of thissolution using a volumetric pipette, place in a 10-mL volumetric flask, and add water tothe marked line (i.e. volume up to 10 mL). Measure 4 mL of this solution using a
volumetric pipette, place in a 50 mL volumetric flask, add a small amount of nitric acid (1→ 150), and then add water to the marked line (i.e. volume up to 50 mL) to prepare thestandard solution. When the sample solution and the standard solution are measured byatomic absorption spectrometry (flameless method (electric heating method)) under theconditions below, the absorbance of the sample solution shall be no more than theabsorbance of the standard solution (no more than 20 μg/g).
Light source lamp: Aluminum hollow cathode lamp
Analysis line wavelength: 309.3 nm

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Provisional Translation from Japanese Original

Drying temperature: 140 °C
Incineration temperature: 900 °C
Atomization temperature: 2,600 °C
Water content No more than 5.0 % (direct titration)
Assay: Weigh approximately 0.20 g of this product to the digit of 0.001 g, record the value, add
methanol to dissolve, place in a 200 mL volumetric flask, and further add methanol to themarked line (i.e. volume up to 10 mL) to prepare the sample stock solution. Measure 5 mL ofthis sample stock solution using a volumetric pipette, place in a 50 mL volumetric flask, add
methanol to the marked line (i.e. volume up to 50 mL), filter with a membrane filter (0.45μm), and serve the filtrate as the sample solution. Separately, weigh approximately 0.050 g of
25-hydroxycholecalciferol reference standard to the digit of 0.0001 g, record the value, add
methanol to dissolve, place in a 50 mL volumetric flask, and further add methanol to themarked line (i.e. volume up to 50 mL). Measure 5 mL of this solution using a volumetricpipette, place in a 50 mL volumetric flask, and add methanol to the marked line (i.e. volume
up to 50 mL) to prepare the standard stock solution. Filter this standard stock solution with amembrane filter (0.45 μm) to prepare a standard solution. For 20 μL of the sample solution
and standard solution, perform testing by liquid chromatography under the conditions below.
Calculate the peak area of 25-hydroxycholecalciferol from the chromatogram obtained.
Content of 25-hydroxycholecalciferol (C27H44O2ꞏH2O)(%) =

AT
AS

×

WS
WT

× p × 400

P : Purity of 25-hydroxycholecalciferol reference standard
WT: Weight of this product (mg)

WS : Weight of 25-hydroxycholecalciferol reference standard (mg)
AT: Peak area of 25-hydroxycholecalciferol in the sample solution
AS : Peak area of 25-hydroxycholecalciferol in the standard solution
Operating condition
Detector: Ultraviolet spectrophotometer (measuring wavelength: 270 nm)
Column: Fill a stainless steel tube of 4.6 mm in inner diameter and 150 mm in length withoctadecylsilyl silica gel for liquid chromatography of 5 μm in particle diameter.
Column temperature: A constant temperature around 28 °C
Mobile phase: Methanol-acetonitrile-water mixture solution (55:22:23)
Flow rate: Approximately 1.0 mL per minute(b) Standard of manufacturing method
Shall be manufactured by aerobically culturing cholesta-5,7,24-trienol production
recombinants hosted by bacterial strains belonging to Saccharomyces cerevisiae, heat

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treating the cultured solution when the culturing is complete to separate cholesta-5,7,24trienol, and by chemically treating (i.e. ultraviolet light irradiation).
(c) Standard of storage method
Shall be placed in a light-resistant sealed container, air replaced with nitrogen, and stored
in a cool place.
B. Preparation (Part 1)(a) Compositional standards
This product is powder in which excipient substances are mixed into source material for
manufacturing 25-hydroxycholecalciferol.
Content: When quantified, this product contains 25-hydroxycholecalciferol (C27H44O2·H2O)
at an amount equivalent to 90~120% of the declared content.
Confirmation test:
i. When 100 μL of a sample solution prepared by a quantification method and a standard
solution is tested by liquid chromatography under the operating condition of thequantification method, the retention time pertaining to the peak of 25hydroxycholecalciferol (C27H44O2·H2O) obtained from the sample solution is the same asthat obtained from the standard solution.
ii. Spot 10 μL each of the sample stock solution prepared by a quantification method andthe standard solution to a thin layer plate prepared using silica gel for thin layer
chromatography containing fluorescent material. Develop for about 12 cm using an nhexane - ethyl acetate mixed solution (1:1) as the developing solvent, and dry the thinlayer plate in air. When an ultraviolet light (main wavelength 254 nm) is irradiated, theRf value of the spot of 25-hydroxycholecalciferol obtained from the sample stock
solution shall be the same as that obtained from the standard stock solution.
Loss on drying: No more than 8.0 % (1 g, 105 °C, 4 hours)
Assay: Weigh approximately 0.35 g of this product to the digit of 0.001 g, record the value,place in a 100 mL volumetric flask, add 15 mL of dimethyl sulfoxide, and sonicate until
the powder becomes invisible. When the solution becomes semi-transparent, add ethyl
acetate to the marked line (i.e. volume up to 100 mL), and allow to stand for 5 minutes toprepare the sample stock solution. Measure 3 mL of the supernatant of this sample stock
solution using a volumetric pipette, place in a 100 mL volumetric flask, add isopropanolethyl acetate-isooctane mixed solution (1:30:69) to the marked line (i.e. volume up to 100mL), and filter with a membrane filter (0.45 μm) to prepare the sample solution.
Separately, weigh approximately 0.020 g of 25-hydroxycholecalciferol reference standard
to the digit of 0.0001 g, record the value, place in a 2000 mL volumetric flask, add 15 mLof methanol, shake for 20 minutes to dissolve, and add ethyl acetate to the marked line (i.e.

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volume up to 200 mL) to prepare the standard stock solution. Measure 3 mL of thisstandard stock solution using a volumetric pipette, place in a 200 mL volumetric flask, add
isopropanol-ethyl acetate-isooctane mixed solution (1:30:69) to the marked line (i.e.
volume up to 200 mL), filter with a membrane filter (0.45 μm), and serve the filtrate as astandard solution. Separately, allow 10 mL of the standard solution to stand at 40°C
overnight or at room temperature for 3~4 days to generate pre-25-hydroxycholecalciferol,filter with a membrane filter (0.45 μm), and serve the filtrate as a separating solution forchecking the retention time of pre-25-hydroxycholecalciferol and for column selection.
For 100 μL of the sample solution, standard solution and separating solution, performtesting by liquid chromatography under the conditions below. Checking of 25hydroxycholecalciferol and pre-25-hydroxycholecalciferol in a sample shall be carried out
by checking the fact that the retention time of the standard solution is the same as that of
the separating solution or the fact that the peak does not broaden when the standard
solution or separating solution is added.
25-hydroxycholecalciferol
AT1 +AT2 ×2.21
WS
=
×
× 25
(C
Content of 27H44O2ꞏH2O)(%)
AS
WT

WT: Weight of this product (mg)

WS : Weight of 25-hydroxycholecalciferol reference standard (mg)
AT1: Peak area of 25-hydroxycholecalciferol in the sample solution
AT2: Peak area of pre-25-hydroxycholecalciferol in the sample solution
AS : Peak area of 25-hydroxycholecalciferol in the standard solution
Operating condition
Detector: Ultraviolet spectrophotometer (measuring wavelength: 260 nm)
Column: Fill a stainless steel tube of 4. 6 mm in inner diameter and 150 mm in lengthwith silica gel for liquid chromatography of 5 μm in particle diameter.
Column temperature: Room temperature
Mobile phase: Isopropanol-ethyl acetate-isooctane mixed solution (1:10:89)
Flow rate: Approximately 1.5 mL per minute
Column selection: When the operation above is applied to 100 μL of this separating
solution, the column to be used shall elute in the order of 25-hydroxycholecalciferol
and then pre-25-hydroxycholecalciferol and the degree of separation shall be no lessthan 1.5.
(b) Standard of storage method
Shall be stored in a light-resistant airtight container.

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Provisional Translation from Japanese Original(81) Ferrous Fumarate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 96.5 % or more of ferrous fumarate(C4H2FeO4).
Physical and chemical properties:
i. This product is red-yellow or red-brown powder with no odor.
ii. This product is hard to dissolve in water and hardly dissolves in ethanol.
iii. This product is decomposed by hydrochloric acid to deposit fumaric acid.
Confirmation test:
i. 0.5 g (0.45~0.54 g) of this product is added with 100 mL of 0.1 mol/L hydrochloric
acid test solution, dissolved by warming, and filtered. The filtrate gives the qualitative
reaction i. of ferrous salt.
ii. 2.0 g (1.95~2.04 g) of this product is added with 25 mL of dilute hydrochloric acid,warmed on a water bath for 15 minutes, allowed to stand, and filtered. The residue iswashed several times using hydrochloric acid (1 → 100), and dried at 100 °C for 30
minutes. 0.05 g (0.045~0.054 g) of it is weighed, added with 3 mg (2.5~3.4 mg) and 1
mL of sulfuric acid, shaken up, and heated at 120~130 °C for 5 minutes. It is allowed
to cool, added with water to make 5 ml, gradually added with sodium hydroxide
solution (2 → 5) by dropping while cooling to make it alkaline, and added with water
to make 10 mL. When this solution is irradiated with ultraviolet light (365 nm), it
emits green fluorescence.
Purity test:i. Lead: When 1.0 g (0.95~1.04 g) of this product is weighed and subjected to the leadtest method (Method No. 1 of atomic absorption spectrophotometry) its amount shallbe 10 mg/kg or less.
ii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed, added with 15 mL of
dilute sulfuric acid, gently heated. When the sample solution is tested for arsenic by
the method using device A, the color of the absorbing solution shall not be darker than
the standard color (5 mg/kg or less)
Loss on drying: Less than 1.0 % (1 g, 105 °C, 2 hours)
Assay: 0.5 g of this product is weighed to the digits of 0.001 g and the value is recorded. It
is placed in a 300 mL stoppered flask, added with 25 mL of hydrochloric acid (2 → 5),boiled, added with a solution of 5.6 g (5.55~5.64 g) of stannous chloride in 50 mL ofhydrochloric acid (3 → 10) by dropping until the color of yellow disappears, and addedwith 2 more drops. The resulting solution is quenched down to room temperature, added

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with 10 mL of mercuric chloride solution (1 → 20), allowed to stand for 5 minutes, addedwith 200 mL of water, 25 mL of sulfuric acid (1 → 2) and 4 mL of phosphoric acid, andtitrated with 0.1 mol/L ceric ammonium sulfate solution (indicator: 2 drops of ophenanthroline test solution).
0.1 mol/L ceric ammonium sulfate solution 1 mL = 16.99 mg C4H2FeO4(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of ferrous fumarate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ferrous fumarate
is applied mutatis mutandis.

(82) Peptide Zinc
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 5.5~8.5 % of nitrogen (N) and10.0~13.0 % of zinc (Zn) at dry-matter conversion.
Physical and chemical properties:
i. This product is pale yellow to brown powder with a specific odor.
ii. This product hardly dissolves in water or ethanol.
Confirmation test:
i. 0.5 g (0.45~0.54 g) of this product is weighed, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution, and warmed in a water bath at 60 °C for 3 minutes
while gently shaking. It is allowed to cool, added with 25 mL of 0.5 mol/Lhydrochloric acid test solution and shaken up. 10 mL of this solution is measured,
added with 10 mL of trichloroacetic acid solution (1 → 10), shaken up, and allowed tostand for 10 minutes. This solution is filtered using filter paper and the filtrate is used
as a sample solution. 1 mL of the sample solution is measured, added with 1 mL ofpyridine, 1 mL of ascorbic acid solution (1 → 2,000), 1 mL of ninhydrin solution (1 →100), and heated on a water bath for 2 minutes. The resulting solution is blue-violet.

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Provisional Translation from Japanese Originalii. 1 mL of the sample solution obtained in i. is measured and added with 1~2 drops ofpyridine and 1 mL of potassium thiocyanate test solution. White precipitation isformed in the resulting solution.
iii. When the infrared absorption spectrum of this product is measured using thepotassium bromide disk method of the infrared absorption spectroscopy, absorptions
are observed at around wavelengths around 3,300~3,600 cm−1 and 1,640~1,660 cm−1.
Purity test:i. Lead: 1.0 g (0.95~1.04 g) of this product is weighed, added with 3 mL of nitric acidand 5 mL of perchloric acid, evaporated to dryness, allowed to cool, added with 5 mLof dilute hydrochloric acid, and warmed on a water bath to dissolve. It is allowed tocool, added with 5 mL of water, mixed, and filtered using filter paper. The residue iswashed with 5 mL of water. The filtrate and the washings are mixed, transferred to a
25 mL volumetric flask, and added with water to the graduation line to make 25 mL.
This is used as a sample solution. Separately, 4 mL of lead standard solution ismeasured using a volumetric pipette, transferred to a 50 mL volumetric flask, andadded with water to the graduation line to make 50 mL. 10 mL of this solution ismeasured using a volumetric pipette, transferred to a 25 mL volumetric flask, andadded with 5 mL of dilute hydrochloric acid and water to the graduation line to make
25 mL. This is used as a standard solution. When the sample and standard solutions aremeasured by the atomic absorption spectrophotometry (flame type) under thefollowing conditions, the absorbance of the sample solution shall be lower than that of
the standard solution (20 mg/kg or less).
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Lead hollow cathode lamp
Wavelength: 217.0 nm
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less)
Loss on drying: Less than 12.0 % (0.5 g, 110 °C, 1 hours)
Rate of hydrolysis: Approximately 1 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is added with 30 mL of 0.5 mol/L hydrochloric acid test solution,
warmed in a water bath at 60 °C for 30 minutes with occasional shaking. It is allowed tocool, and transferred to a 50 mL volumetric flask, added with 10 mL of trichloroacetic
acid solution (1 → 10), vigorously shaken up, added with 0.5 mol/L hydrochloric acid416

Provisional Translation from Japanese Original

test solution to the graduation line to make 50 mL. Approximately 20 mL of this solution
is measured, centrifuged for 10 minutes at 3,000 rpm. 5 mL of the supernatant fluid
obtained is measured using a volumetric pipette, transferred to a 25 mL volumetric flask,
and added with water to the graduation line to make 25 mL. 4 mL of this solution ismeasured using a volumetric pipette, transferred to a Kjeldahl flask, added with 10 mL of
sulfuric acid along the sulfuric acid of the flask. The test is hereinafter performed by thenitrogen determination method (Semimicro-Kjeldahl Method). Based on the rate ofhydrolysis obtained here (A%), and total nitrogen (B%) obtained in the section of Assay,
the rate of hydrolysis determined by the following equation shall be 30 % or more.
Rate of hydrolysis (%) =

A
× 100
B

Assay:
i. Total nitrogen: Approximately 0.03 g of this product is weighed to the digits of 0.1 mg
and the value is recorded. It is placed in a Kjeldahl flask and the test is hereinafter
performed by the nitrogen determination method (Semimicro-Kjeldahl Method).
0.005 mol/L sulfuric acid 1 mL = 0.1401 mg N
ii. Zinc: Approximately 0.2 g of this product is measured to the digits of 0.001 g and thevalue is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitric acid and10 mL of perchloric acid and heated until the residue reaches approximately 2 mL. It isallowed to cool, added with 2 mL of hydrochloric acid and heated on a water bath todissolve. It is allowed to cool, transferred to a 100 mL volumetric flask, and addedwith more water to the graduation line to make 100 mL. This solution is filtered using
filter paper. 10 mL of the first filtrate is discarded, 10 mL of the next filtrate ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, andadded with water to make 100 mL. Then, 5 mL of this solution is measured using a
volumetric pipette, transferred to a 100 mL volumetric flask and added with water tothe graduation line to make 100 mL. This is used as a sample solution. Separately, 5
mL of nitric acid and 10 mL of perchloric acid are placed in a Kjeldahl flask andheated until the residue reaches approximately 2 mL. It is allowed to cool, added with
2 mL of hydrochloric acid, transferred to a 100 mL volumetric flask, and added withwater to the graduation line to make 100 mL. 10 mL of this solution is measured using
a volumetric pipette, transferred to a 100 mL volumetric flask, and added with water tothe graduation line to make 100 mL. This is used as a blank test stock solution. 5 mLof this solution is measured using a volumetric pipette, transferred to a 100 mL
volumetric flask, and added with water to the graduation line to make 100 mL. This isused as a blank test solution. Separately, 5, 10, 15 and 20 mL each of zinc standard

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Provisional Translation from Japanese Original

solutions is measured using a volumetric pipette, transferred to a 100 mL volumetricflask, 5 mL of the blank test stock solution is added using a volumetric pipette, andadded with water to the graduation line to make 100 mL. The resulting solutions arecalled the standard solution 1, 2, 3 and 4, respectively. The sample solution, thestandard solutions 1, 2, 3 and 4, and the blank test solution are determined by theatomic absorption spectrophotometry (flame type) under the following conditions. A
calibration curve is prepared from the absorbances of the standard solution 1, 2, 3 and4, and the blank test solution and the content of zinc in the sample is calculated.
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Zinc hollow cathode lamp
Wavelength: 213.9 nm
Content of zinc (%)

Concentration of zine in the sample solution obtained=

from the calibration curve (µg/mL)
× 2,000
collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of peptide zinc areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of peptide zinc is
applied mutatis mutandis.

(83) Peptide Iron
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 7.0~10.5 % of nitrogen (N) and10.0~13.0 % of iron (Fe) at dry-matter conversion.
Physical and chemical properties:
i. This product is brown to greenish brown powder or particles with a specific odor.
ii. This product hardly dissolves in water and ethanol.

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Provisional Translation from Japanese OriginalConfirmation test:
i. This product is powdered as appropriate. 0.05 g (0.045~0.054 g) of this product isweighed, added with 25 mL of 0.5 mol/L hydrochloric acid test solution, warmed in awater bath at 60 °C for 3 minutes while gently shaking. It is allowed to cool, addedwith 25 mL of 0.5 mol/L hydrochloric acid test solution, and shaken up. 10 mL of thissolution is measured, added with 10 mL of trichloroacetic acid solution (1 → 10),shaken up, and allowed to stand for 10 minutes. This solution is filtered by filer paper
and the filtrate is used as a sample solution. 1 mL of the sample solution is measured,
added with 1 mL of pyridine, 1 mL of ascorbic acid solution (1 → 2,000) and 1 mL ofninhydrin solution (1 → 100), and heated on a water bath for 2 minutes. The resulting
solution is blue-violet.
ii. 1 mL of the sample solution obtained in i. is measured, added with 1 mL ofhydroxylamine hydrochloride solution (1 → 50), allowed to stand for 10 minutes,
dissolved with 2 mL of a solution prepared by the way in which 0.25 g of ophenanthroline is dissolved with pH 5.0 acetic acid-sodium acetate buffer to make 100
mL. The resulting solution is red to red-orange.
Purity test:i. Lead: 1.0 g (0.95~1.04 g) of this product is weighed. When lead is tested by the leadtest method (Method No. 1 of the atomic absorption spectrophotometry), the amountof lead shall be 10 mg/kg or less.
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less)
Loss on drying: Less than 13.0 % (0.5 g, 110 °C, 8 hours)
Rate of hydrolysis: This product is powdered as appropriate. Approximately 1 g of thisproduct is weighed to the digits of 0.01 g and the value is recorded. It is added with 30
mL of 0.5 mol/L hydrochloric acid and warmed in a water bath at 60 °C for 30 minutes
while gently shaking. It is allowed to cool, transferred to a 50 mL volumetric flask, addedwith 10 mL of trichloroacetic acid solution (1 → 10), vigorously shaken up, and addedwith 0.5 mol/L hydrochloric acid to the graduation line to make 50 mL. Approximately
20 mL of this solution is measured, and centrifuged for 5 minutes at 4,000 rpm. 5 mL of
the supernatant fluid obtained is measured using a volumetric pipette, transferred to a 25mL volumetric flask and added with water to the graduation line to make 25 mL. 4 mL ofthis solution is measured using a volumetric pipette, transferred to a Kjeldahl flask, andadded with 7 mL of sulfuric acid along the inner wall of the flask. The test is hereinafter

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Provisional Translation from Japanese Original

performed by the nitrogen determination method. Based on the rate of hydrolysis of thedry-matter obtained here (A%), and total nitrogen (B%) obtain in the section of Assay,
the rate of hydrolysis determined by the following equation shall be 30 % or more.
Rate of hydrolysis (%) =

A
× 100
B

Assay:
i. Total nitrogen: This product is powdered as appropriate. Approximately 0.03 g of thisproduct is weighed to the digits of 0.1 mg and the value is recorded. It is placed in a
Kjeldahl flask. The test is hereinafter performed by the nitrogen determination method.
0.005 mol/L sulfuric acid 1 mL = 0.1401 mg N
ii. Iron: This product is powdered as appropriate. Approximately 0.2 g of this product isweighed to the digits of 0.001 g and the value is recorded. It is placed in a Kjeldahl
flask, added with 3 mL of nitric acid and 5 mL of perchloric acid, and heated until theresidual liquid reaches approximately 1 mL (about 2 hours). After cooling, the residualliquid is added with 2 mL of hydrochloric acid and heated on a water bath to dissolve.
It is allowed to cool, transferred to a 100 mL volumetric flask and added with water tothe graduation line to make 100 mL. This solution is filtered by filter paper. 10 mL of
the first filtrate is discarded, 10 mL of the next filtrate is measured using a volumetricpipette, transferred to a 100 mL volumetric flask, and added with water to make 100
mL. Then, 5 mL of this solution is measured using a volumetric pipette, transferred toa 100 mL volumetric flask and added with water to the graduation line to make 100
mL. This is used as a sample solution. Separately, 3 mL of nitric acid and 5 mL ofperchloric acid are placed in a Kjeldahl flask and heated until the residue reaches
approximately 1 mL. It is allowed to cool, added with 2 mL of hydrochloric acid,transferred to a 100 mL volumetric flask, and added with water to the graduation line
to make 100 mL. 10 mL of this solution is measured using a volumetric pipette,
transferred to a 100 mL volumetric flask, and added with water to the graduation line
to make 100 mL. This is used as a blank test stock solution. 5 mL of this solution ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. This is used as a blank test
solution. Separately, 5, 10, 15 and 20 mL each of iron standard solutions is measured
using a volumetric pipette, transferred to a 100 mL volumetric flask, 5 mL of the blanktest stock solution is added using a volumetric pipette, and added with water to thegraduation line to make 100 mL. The resulting solutions are called the standard
solution 1, 2, 3 and 4, respectively. The sample solution, the standard solutions 1, 2, 3
and 4, and the blank test solution are determined by the atomic absorption

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Provisional Translation from Japanese Original

spectrophotometry (flame type) under the following conditions. A calibration curve isprepared from the absorbances of the standard solution 1, 2, 3 and 4 and the content ofiron in the sample (mg/kg) is calculated.
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Iron hollow cathode lamp
Wavelength: 248.3 nm
Content of iron (%)=

Amount of iron from the calibration curve (mg/kg)
× 2,000
collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of peptide iron are
applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of peptide iron isapplied mutatis mutandis.

(84) Peptide Copper
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 5.5~8.5 % of nitrogen (N) and10.0~13.0 % of copper (Cu) at dry-matter conversion.
Physical and chemical properties:
i. This product is blue-green powder with a specific odor.
ii. This product hardly dissolves in water or ethanol.
Confirmation test:
i. The confirmation test i. of the raw material for manufacturing of peptide zinc is appliedmutatis mutandis.
ii. When 1 mL of the sample solution obtained in i. is measured, added with 1 mL ofpotassium ferrocyanide test solution, red-brown precipitation is formed in the resulting
solution.

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Provisional Translation from Japanese Originaliii. When the infrared absorption spectrum of this product is measured using thepotassium bromide disk method of the infrared absorption spectroscopy, absorptions
are observed at around wavelengths around 3,300~3,600 cm−1 and 1,630~1,650 cm−1.
Purity test:i. Lead: The purity test i. of the raw material for manufacturing of peptide zinc is appliedmutatis mutandis (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less)
Loss on drying: Less than 12.0 % (0.5 g, 110 °C, 1 hour)
Assay:
i. Total nitrogen: Approximately 0.03 g of this product is weighed to the digits of 0.1 mg
and the value is recorded. It is placed in a Kjeldahl flask. The test is hereinafter
performed by the nitrogen determination method.
0.005 mol/L sulfuric acid 1 mL = 0.1401 mg N
ii. Copper: Approximately 0.2 g of this product is weighed to the digits of 0.001 g andthe value is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitric acidand 10 mL of perchloric acid and heated until the residual liquid reaches
approximately 2 mL. After cooling, it is added with 2 mL of hydrochloric acid, and isheated on a water bath to dissolve. It is allowed to cool, transferred to a 100 mL
volumetric flask, and added with water to the graduation line to make 100 mL. Thissolution is filtered by filter paper. 10 mL of the first filtrate is discarded, 10 mL of thenext filtrate is measured using a volumetric pipette, transferred to a 100 mL volumetricflask, and added with water to make 100 mL. Then, 5 mL of this solution is measured
using a volumetric pipette, transferred to a 100 mL volumetric flask and added withwater to the graduation line to make 100 mL. This is used as a sample solution.
Separately, 5 mL of nitric acid and 10 mL of perchloric acid are placed in a Kjeldahl
flask and heated until the residue reaches approximately 2 mL. It is allowed to cool,transferred to a 100 mL volumetric flask, added with 2 mL of hydrochloric acid, andadded with water to the graduation line to make 100 mL. 10 mL of this solution ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. This is used as a blank test
stock solution. 5 mL of this solution is measured using a volumetric pipette,
transferred to a 100 mL volumetric flask, and added with water to the graduation line
to make 100 mL. This is used as a blank test solution. Separately, 2, 4, 6 and 8 mL

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Provisional Translation from Japanese Original

each of copper standard solutions is measured using a volumetric pipette, transferred toa 100 mL volumetric flask, 5 mL of the blank test stock solution is added using a
volumetric pipette, and added with water to the graduation line to make 100 mL. The
resulting solutions are called the standard solution 1, 2, 3 and 4, respectively. The
sample solution, the standard solutions 1, 2, 3 and 4, and the blank test solution aredetermined by the atomic absorption spectrophotometry (flame type) under thefollowing conditions. A calibration curve is prepared from the absorbances of thestandard solution 1, 2, 3 and 4 and the blank test solution and the content of copper inthe sample is calculated.
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Copper hollow cathode lamp
Wavelength: 324.7 nm
Content of copper (%)
Concentration of copper in the sample solution obtained
from the calibration curve (µg/mL)=
× 2,000
collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of peptide copper are
applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of peptide copper
is applied mutatis mutandis.

(85) Peptide Manganese
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 5.5~8.5 % of nitrogen (N) and10.0~13.0 % of manganese (Mn) at dry-matter conversion.
Physical and chemical properties:
i. This product is pale yellow to brown powder with a specific odor.
ii. This product hardly dissolves in water or ethanol.

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Provisional Translation from Japanese OriginalConfirmation test:
i. The confirmation test i. of the raw material for manufacturing of peptide zinc is appliedmutatis mutandis.
ii. When 1 mL of the sample solution obtained in i. is measured, added with 5 mL ofammonia test solution and 5 mL of silver nitrate test solution, white precipitation isformed in the solution. The generated precipitation gradually turns black.
iii. When the infrared absorption spectrum of this product is measured using thepotassium bromide disk method of the infrared absorption spectroscopy, absorptions
are observed at around wavelengths around 3,300~3,600 cm−1 and 1,630~1,650 cm−1.
Purity test:i. Lead: The purity test i. of the raw material for manufacturing of peptide zinc is appliedmutatis mutandis (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less)
Loss on drying: Less than 12.0 % (0.5 g, 110 °C, 1 hour)
Rate of hydrolysis: The rate of hydrolysis of the raw material for manufacturing of peptide
zinc is applied mutatis mutandis
Assay:
i. Total nitrogen: Approximately 0.03 g of this product is weighed to the digits of 0.1 mg
and the value is recorded. It is placed in a Kjeldahl flask. The test is hereinafter
performed by the nitrogen determination method.
0.005 mol/L sulfuric acid 1 mL = 0.1401 mg N
ii. Manganese: Approximately 0.2 g of this product is weighed to the digits of 0.001 g
and the value is recorded. It is placed in a Kjeldahl flask, added with 5 mL of nitricacid and 10 mL of perchloric acid and heated until the residual liquid reaches
approximately 2 mL. After cooling, it is added with 2 mL of hydrochloric acid, andheated on a water bath to dissolve. It is allowed to cool, transferred to a 100 mL
volumetric flask, and added with water to the graduation line to make 100 mL. Thissolution is filtered by filter paper. 10 mL of the first filtrate is discarded, 10 mL of thenext filtrate is measured using a volumetric pipette, transferred to a 100 mL volumetricflask, and added with water to make 100 mL. Then, 5 mL of this solution is measured
using a volumetric pipette, transferred to a 100 mL volumetric flask and added withwater to the graduation line to make 100 mL. This is used as a sample solution.
Separately, 5 mL of nitric acid and 10 mL of perchloric acid are placed in a Kjeldahl

424

Provisional Translation from Japanese Original

flask and heated until the residue reaches approximately 2 mL. It is allowed to cool,transferred to a 100 mL volumetric flask, added with 2 mL of hydrochloric acid, andadded with water to the graduation line to make 100 mL. 10 mL of this solution ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. This is used as a blank test
stock solution. 5 mL of this solution is measured using a volumetric pipette,
transferred to a 100 mL volumetric flask, and added with water to the graduation line
to make 100 mL. This is used as a blank test solution. Separately, 5, 10, 15 and 20 mL
each of manganese standard solutions is measured using a volumetric pipette,
transferred to a 100 mL volumetric flask, 5 mL of the blank test stock solution is addedusing a volumetric pipette, and added with water to the graduation line to make 100
mL. The resulting solutions are called the standard solution 1, 2, 3 and 4, respectively.
The sample solution, the standard solutions 1, 2, 3 and 4, and the blank test solution
are determined by the atomic absorption spectrophotometry (flame type) under thefollowing conditions. A calibration curve is prepared from the absorbances of thestandard solution 1, 2, 3 and 4 and the blank test solution and the content ofmanganese in the sample is calculated.
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Manganese hollow cathode lamp
Wavelength: 279.5 nm
Content of manganese (%)
Concentration of manganese in the sample solution obtained
from the calibration curve (µg/mL)=
× 2,000
collected amount of the sample (mg)(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of peptide manganeseare applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of peptide
manganese is applied mutatis mutandis.

425

Provisional Translation from Japanese Original(86) DL-methionine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 4-hour drying at 105 °C, it contains
98.5 % or more of DL-methionine (C5H11NO2S)
Physical and chemical properties:
i. This product is white to pale-yellow crystals or crystalline powder with a specific odor
and with slight sweetness.
ii. This product is hard to lightly dissolve in water, hard to extremely dissolve in ethanol,and hardly dissolves in ether.
iii. This product dissolves in dilute hydrochloric acid or 1 mol/L sodium hydroxide test
solution.
iv. The pH of a solution of this product in water (1 → 100) is 5.2 to 6.1
v. A solution of this product in water (1 → 100) does not have an optical rotation.
Confirmation test:
i. 25 mg (24.5~25.4 mg) of this product is added with 1 mL of sulfuric acid saturated
with anhydrous copper sulfate. The resulting solution is yellow.
ii. 5 mg (4.5~5.4 mg) of this product is dissolved with 5 mL of water, added with 2 mLof 1 mol/L sodium hydroxide test solution, shaken up well, added with 0.3 mL of
sodium nitroprusside test solution, shaken up well again, allowed to stand at 35~40 °C
for 10 minutes, cooled with ice for 2 minutes, added with 2 mL of dilute hydrochloric
acid and shaken up. The resulting solution is red.
Purity test:
i. Clarity and color of solution: 0.5 g (0.45~0.54 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and almost clear.
ii. Chloride: When chloride is tested using a sample solution prepared with 0.09 g(0.085~0.094 g) of this product by the chloride test method and a control solution
prepared with 0.5 mL of 0.01 mol/L hydrochloric acid, the turbidity of the sample
solution shall not be higher than that of the control solution (0.20 % or less).
iii. Sulfate: When sulfate is tested using a sample solution prepared with 0.08 g(0.075~0.084 g) of this product by the sulfate test method and a control solution
prepared with 0.5 mL of 0.005 mol/L sulfuric acid, the turbidity of the sample solution
shall not be higher than that of the control solution (0.30 % or less).
iv. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using

426

Provisional Translation from Japanese Original

these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is placed in a 100 mL decomposition flask,
and is added with 5 mL of nitric acid and 2 mL of sulfuric acid. A small funnel isplaced on the mouth of the flask and the solution is carefully heated until white smoke
emerges. It is allowed to cool, added with 2 mL each of nitric acid twice, heated,added with 2 mL each of hydrogen peroxide solution several times, and heated until
the solution reaches colorless to slightly yellow. It is allowed to cool, added with 2 mLof saturated ammonium oxalate and again heated until white smoke emerges. It isallowed to cool and added with water to make 5 mL. This is used as a sample solution.
When arsenic is tested by the method using device A, the color of the absorbing
solution shall not be darker than the standard color (2 mg/kg or less).
Loss on drying: Less than 0.5 % (1 g 105 °C, 4 hours)
Ignition residue: Less than 0.5 % (1 g)Assay: This product is dried at 105 °C for 4 hours, approximately 0.3 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is placed in a stoppered flask, addedwith 100 mL of water, 5 g (4.5~5.4 g) of dibasic potassium phosphate, 2 g (1.5~2.4 g) ofpotassium dihydrogenphosphate and 2 g (1.5~2.4 g) of potassium iodide, and shaken up
to dissolve. It is added with 50 mL of 0.1 mol/L iodine solution using a volumetricpipetted, tightly stoppered, shaken up well, and allowed to stand for 30 minutes, and an
excess amount of iodide is titrated with 0.1 mol/L sodium thiosulfate solution (indicator:
1 mL of starch test solution). A blank test is performed in the same manner.
0.1 mol/L iodine solution 1 mL = 7.461 mg C5H11NO2S(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of DL-methionine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of DL-methionine
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is particles in which the raw material for manufacturing of DL-methionine is
added with ethyl cellulose and sodium stearate as appropriate, and mixed with fillers.

427

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains DL-methionine (C5H11NO2S)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 25 mg of
the raw material for manufacturing of DL-methionine is weighed, and the confirmation
test i. for the raw material for manufacturing of DL-methionine is hereinafter appliedmutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 5 mg of
the raw material for manufacturing of DL-methionine is weighed, added with 1 mL ofa mixed solution of n-hexane and isopropanol (1:1), and shaken up. This solution isadded with 5 mL of water, shaken up, and allowed to stand. The aqueous layer isfiltered and the confirmation test ii. for the raw material for manufacturing of DLmethionine is hereinafter applied to the filtrate mutatis mutandis.
A preparation containing fatty acid calcium is powdered. Then, according to theamount of this product on the label, the amount containing 5 mg of the raw material
for manufacturing of DL-methionine is weighed, added with 5 mL of water, shaken
up, and the confirmation test ii. for the raw material for manufacturing of DLmethionine is hereinafter applied mutatis mutandis.
Assay: Method No. 1 is used for a preparation containing fatty acid calcium and Method
No. 2 is used for the others.
Method No. 1: This product is powdered, the amount of it containing approximately 0.03
g of DL-methionine (C5H11NO2S) is weighed to three significant digits and the value isrecorded. It is added with 70 mL of hydrochloric acid (9 → 250), heated at
approximately 90 °C for 30 minutes while stirring to dissolve, allowed to cool, andfiltered. 100 mL of the filtrate is transferred to a 100 mL volumetric flask, added withhydrochloric acid (9 → 250) to the graduation line to make 100 mL. 2 mL of thissolution is measured using a volumetric pipette and added with 2 mL of hydrochloric
acid (9 → 250) and 12 mL of water using a volumetric pipette. This is used as asample solution. Separately, 0.030 g of dried DL-methionine for assay is weighed tothe digits of 0.1 mg and the value is recorded. It is dissolved with hydrochloric acid (9→ 250), transferred to a 100 mL volumetric flask, and added with more hydrochloric
acid (9 → 250) to the graduation line to make 100 mL. 2 mL of this solution ismeasured using a volumetric pipette and added with 2 mL of hydrochloric acid (9 →250) and 12 mL of water using a volumetric pipette. This is used as a standard
solution. Separately, 2 mL of hydrochloric acid (9 → 250) is measured using a
volumetric pipette and added with 6 mL of water using a volumetric pipette. This is428

Provisional Translation from Japanese Originalused as a blank test solution. Then, 2 mL each of the sample, standard and blank test
solutions is measured using a volumetric pipette, transferred to a 25 mL volumetricflask, added with 1 mL of newly prepared ninhydrin ascorbate test solution and 1 mLof pyridine using a volumetric pipette, and shaken up. Then, it is heated in a water bathfor 25 minutes, allowed to cool, and added with water to the graduation line to make
25 ml. The absorbances, the sample and standard solutions, AT and AS respectively, ata wavelength 570 nm are measured using the blank test solution as a control solution.
Amount of DL-methionine (C5H11NO2S) (mg)
= Amount of DL-methionine for assay (mg) ×

AT
AS

Method No. 2: This product is powdered, the amount of it containing approximately 0.03
g of DL-methionine (C5H11NO2S) is weighed to three significant digits, and the value
is recorded. It is added with 15 mL of a mixed solution of n-hexane and isopropanol(1:1), stirred to dissolve, added with 70 mL of hydrochloric acid (9 → 250) andextracted while vigorously stirring. This solution is transferred to a separatory funnel
while filtering with absorbent cotton and the container is washed with 5 mL of a mixed
solution of n-hexane and isopropanol (1:1) and 15 mL of hydrochloric acid (9 → 250).
The washings are poured into the absorbent cotton and these filtrates are mixed. The
resulting solution is shaken up and the aqueous layer is transferred to a 100 mL
volumetric flask and added with hydrochloric acid (9 → 250) to the graduation line to
make 100 mL. 2 mL of this solution is measured using a volumetric pipette, addedwith 2 mL of hydrochloric acid (9 → 250) and 12 mL of water using a volumetricpipette. This is used as a sample solution and Method No. 1 is hereinafter appliedmutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of DL-methionine
is applied mutatis mutandis.
(c) Standard of the label
As for a preparation for cattle, which is coated for degradation less in the rumen but morein the abomasum, the following words shall be written on the immediate container or theimmediate wrapper of this product.
Precautions for use: Since this product is covered for degradation less in the rumen of thecattle but more in the abomasum, use it with attention to the amino-acid balance.

429

Provisional Translation from Japanese Original(87) L-methionine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 4-hour drying at 105 °C, it contains
98.5 % or more of L-methionine (C5H11NO2S)
Physical and chemical properties:
i. This product is white to pale-yellow crystals or crystalline powder.
ii. This product is easy to lightly dissolve in water, and hardly dissolves in ethanol andether.
iii. This product dissolves in dilute hydrochloric acid or 1 mol/L sodium hydroxide test
solution.
iv. The pH of a solution of this product in water (1 → 100) is 5.2 to 6.1
Confirmation test:
Confirmation test of the raw material for manufacturing of DL-methionine are appliedmutatis mutandis.
Purity test:i. Specific rotation: This product is dried at 105 °C for 4 hours. Approximately 1 g of it isweighed to the digits of 0.01 g and the value is recorded. It is dissolved in hydrochloric
acid test solution, 6 mol/L to make 50 mL and as appropriate filtered. The rotation ofthis solution measured shall be [α]25
D = +21.1 ~ +25.1°.

ⅱ. Clarity and color of solution: Purity test i of the raw material for manufacturing of DLmethionine are applied mutatis mutandis.
ⅲ. Chloride: Purity test ii of the raw material for manufacturing of DL-methionine areapplied mutatis mutandis.
ⅳ. Sulfate: Purity test iii of the raw material for manufacturing of DL-methionine areapplied mutatis mutandis.
ⅴ. Lead: When 0.5 g (0.45~0.54 g) of this product is weighed and subjected to the leadtest method (Method No. 1 of atomic absorption spectrophotometry), the content oflead shall be 20 mg/kg or less.
ⅵ. Arsenic: Purity test v of the raw material for manufacturing of DL-methionine areapplied mutatis mutandis.
Loss on drying: Less than 0.5 % (1 g 105 °C, 4 hours)
Ignition residue: Less than 0.5 % (1 g)Assay: This product is dried at 105 °C for 4 hours, approximately 0.3 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is placed in a stoppered flask, addedwith 100 mL of water, 5 g (4.5~5.4 g) of dibasic potassium phosphate, 2 g (1.5~2.4 g) of430

Provisional Translation from Japanese Original

potassium dihydrogenphosphate and 2 g (1.5~2.4 g) of potassium iodide, and shaken up
to dissolve. It is added with 50 mL of 0.1 mol/L iodine solution using a volumetricpipetted, tightly stoppered, shaken up well, and allowed to stand for 30 minutes, and an
excess amount of iodide is titrated with 0.1 mol/L sodium thiosulfate solution (indicator:
1 mL of starch test solution). A blank test is performed in the same manner.
0.1 mol/L iodine solution 1 mL = 7.461 mg C5H11NO2S(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-methionine areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-methionine isapplied mutatis mutandis.

(88) Menadione dimethylpyrimidinol bisulfite
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 94.0 % or more of menadione
dimethylpymidinol bisulfite (C17H18N2O6S) at dehydration product conversion.
Physical and chemical properties:
i. This product is white to pale yellow-brown crystalline powder with no odor and with aslightly bitter taste.
ii. This product is hard to lightly dissolve in water and hard to extremely dissolve inethanol, acetone, and ether.
Confirmation test:i. 1 mL of a solution of this product in water (1 → 500) is added with 10 mL of ammonia
test solution and 1 mL of isopropanol, shaken up, added with a drop of ethyl
cyanoacetate, and shaken up. The resulting solution is blue-violet.
ii. 5 mL of a solution of this product in water (1 → 500) is added with 5 mL of ethanol
and 5 mL of a solution of sulfanilic acid in 0.2 mol/L sulfuric acid (1.6 → 100),
shaken up, added with 2 mL of sodium nitrite solution (5 → 100), shaken up, andheated on a water bath for 5 minutes. The resulting solution is reddish violet.

431

Provisional Translation from Japanese Original

Purity test:
i. Heavy metals 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
iii. 2-methyl-1, 4-naphthohydroquinone-3-sulfonate: When 0.10 g (0.095~0.104 g) of thisproduct is dissolved with 5 mL of water, and added with 2 drops of o-phenanthroline
test solution, precipitation shall not be generated.
Water content: Less than 1.0 % (1 g)
Ignition residue: Less than 0.20 % (1 g)Assay: Approximately 0.3 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 30 mL of water, added with 30 mL of chloroform, andthen added with 2 mL of 1 mol/L of sodium hydroxide test solution, and within 30
seconds vigorous shaking of the resulting solution is started and then maintained for 15
seconds. The solution is added with 2 mL more of 1 mol/L sodium hydroxide test
solution, within 30 seconds vigorous shaking is started and maintained for 15 seconds,
and again 2 mL of 1 mol/L sodium hydroxide test solution is added, and within 30
seconds vigorous shaking is started and maintained for 90 seconds. Then it is allowed tostand and the chloroform layer is fractionated. The aqueous layer is extracted twice with
30 mL of chloroform each time. The mixture of all chloroform extracts is washed with 20
mL of water, filtered with pre-chloroform-moisturized filter paper, and washed with 5 mLof chloroform. The filtrate and the washings are mixed and transferred to a 200 mL flask,
and the chloroform is evaporated on a water bath. Then, it is evaporated to dryness while
injecting nitrogen at room temperature. The residue is dissolved with 20 mL of glacialacetic acid and 15 mL of dilute hydrochloric acid, added with 3 g (2.5~3.4 g) of zincpowder, attached with a Bunsen valve, frequently shaken up, allowed to stand in a darkplace for an hour, and filtered using a cotton plug. The flask is washed five times with 20
mL each time with newly boiled and cooled water. The filtrate and the washings are
mixed and titrated with 0.1 mol/L ceric ammonium sulfate solution within 30 seconds(indicator: 2 drops of o-phenanthroline test solution). A blank test is performed in thesame manner and corrections are made.

432

Provisional Translation from Japanese Original

0.1 mol/L ceric ammonium sulfate solution 1 mL = 18.92 mg C17H18N2O6S(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of menadione
dimethylpymidinol bisulfite are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of menadione
dimethylpymidinol bisulfite is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof menadione dimethylpymidinol bisulfite and fillers.
Content: When this product is determined, it contains menadione dimethylpymidinol
bisulfite (C17H18N2O6S) corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.03 g of
the raw material for manufacturing of menadione dimethylpyrimidinol bisulfite isweighed, added with 15 mL of water, and for a preparation containing hydrogenatedoils, higher saturated fatty acids, fatty acids, vegetable oil or animal fats added with 5
mL of chloroform, shaken up, and allowed to stand. The aqueous layer is filtered toprepare a sample solution and 1 mL of this solution is measured. The confirmation test
i. of the raw material for manufacturing of menadione dimethylpyrimidinol bisulfite ishereinafter applied mutatis mutandis.
ii. 5 mL of the sample solution in i. is measured and confirmation test ii. of the raw
material for manufacturing of menadione dimethylpyrimidinol bisulfite is hereinafter
applied mutatis mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forthe others.
Method No. 1: The amount of this product containing approximately 0.1 g of menadione
dimethylpyrimidinol bisulfite (C17H18N2O6S) is weighed to three significant digits andthe value is recorded. It is added with 50 mL of chloroform, shaken up, and filteredusing a glass filter (G4). The residue on the filter is washed three times with 20 mL ofchloroform each time. When the chloroform odor on the residue disappears, the433Provisional Translation from Japanese Originalresidue is filtered by extracting with approximately 80 mL of warm water. The filtrateis placed in a 100 mL volumetric flask, allowed to cool, and added with water to thegraduation line to make 100 mL. This is used as a sample solution. Separately,
approximately 0.1 g of menadione dimethylpyrimidinol bisulfite for assay is weighedto the digits of 0.001 g and the value is recorded. It is dissolved with water, transferred
to a 100 mL volumetric flask, added with more water to the graduation line anddissolved to make 100 mL. This is used as a standard solution. 5 mL each of thesample solution is measured into 100 mL brown volumetric flasks T and T' using a
volumetric pipette. Each of them is added with 80 mL of water, mixed well, addedwith 2 mL of a mixed solution of strong ammonia solution and isopropanol (1:1) andshaken up well. Within 30 seconds, the resulting solution in T is added with 3 mL of asolution of ethyl cyanoacetate in isopropanol (3 → 20), shaken up, and then within 30
seconds, added with water to the graduation line to make 100 mL. The resulting
solution in T' is added with water to the graduation line to make 100 mL. Separately, 5mL each of the standard solution is measured into 100 mL brown volumetric flasks S
and S' using a volumetric pipette, and the same procedure as for the sample solution is
performed. They are allowed to stand for 5 minutes. For each solution, absorbances,
AT, AT', As and AS' at the maximum wavelength around 565 nm are measured using
water as a control.
Amount of menadione dimethylpyrimidinol bisulfite (C17H18N2O6S) (mg)

= Amount of menadione dimethylpyrimidinol bisulfite for assay (C17H18N2O6S) (mg)×AT - AT' 1
×
AS - AS' 2

Method No. 2: The amount of this product containing approximately 0.1 g of menadione
dimethylpyrimidinol bisulfite (C17H18N2O6S) is weighed to three significant digits andthe value is recorded. It is added with 60 mL of water, vigorously shaken up, andfiltered. The residue on the paper filter is washed twice with 10 mL of water each time.
The filtrate and the washings are mixed and transferred to a 100 mL volumetric flask
to make 100 mL. This is used as a sample solution and Method No. 1 is hereinafter
applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

434

Provisional Translation from Japanese Original(89) Menadione Sodium Bisulfite
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 93.0 % or more of menadione sodium
bisulfite (C11H8O2 NaHSO3) at dehydration product conversion.
Physical and chemical properties:
i. This product is white to slight brown crystalline powder with no odor.
ii. This product is easy to dissolve in water, hard to dissolve in ethanol, and hardly
dissolves in ether or benzene.
Confirmation test:
i. 0.1 g (0.05~0.14 g) of this product in placed in a separatory funnel, dissolved with 10
mL of water, added with 3 mL of 1 mol/L sodium hydroxide test solution, andextracted twice with 5 mL of chloroform each time. The chloroform extracts are
mixed, filtered with chloroform moisturized filter paper, and evaporated to dryness
while injecting air at room temperature. When the residue is dissolved with 2 mL ofethanol, and again evaporated to dryness, the melting point is 104~107 °C.
ii. When 2 mL of a solution of this product in water (1 → 1,000) is added with 1 mL ofethanol and 1 mL of ammonia test solution, shaken up, and added with 3 drops of ethyl
cyanoacetate by dropping, the solution is purple-blue. When it is added with 1 mL of
sodium hydroxide solution (1 → 3), the color of the solution turns green and thenbrownish yellow.
iii. When 2 mL of a solution of this product in water (1 → 25) is added with 2~3 drops ofdilute hydrochloric acid and heated, the generated gas changes water-moisturized
potassium iodide-starch paper to blue.
Purity test:
i. Heavy metals 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).

435

Provisional Translation from Japanese Originaliii. 2-methyl-1, 4-naphthohydroquinone-3-sulfonate: When 0.10 g (0.095~0.104 g) of thisproduct is dissolved with 5 mL of water, and added with 2 drops of o-phenanthroline
test solution, precipitation shall not be generated.
Water content: 10.0~16.5 % (0.2 g)
Assay: Approximately 0.3 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 20 mL of water, added with 20 mL of chloroform, shaken
up by adding 5 mL of sodium carbonate solution (1 → 2) little by little, and then morevigorously shaken up. The chloroform layer is fractionated. The aqueous layer is
extracted twice with 20 mL of chloroform each time. The mixture of all chloroform
extracts is washed with 10 mL of water, filtered with pre-chloroform-moisturized filter
paper, and washed with chloroform. The filtrate and the washings are mixed andtransferred to a 150 mL flask, and the chloroform is evaporated on a water bath. Then, it
is evaporated to dryness while injecting nitrogen at room temperature. The residue isdissolved with 20 mL of glacial acetic acid and 15 mL of dilute hydrochloric acid, addedwith 3 g (2.5~3.4 g) of zinc powder, attached with a Bunsen valve, repeatedly shaken up,
allowed to stand in a dark place for an hour, and filtered using a cotton plug. The flask iswashed three times with 20 mL of newly boiled and cooled water each time. The filtrate
and the washings are mixed and titrated with 0.1 mol/L ceric ammonium sulfate solution
within 30 seconds (indicator: 2 drops of o-phenanthroline test solution). A blank test is
performed in the same manner and corrections are made.
0.1 mol/L ceric ammonium sulfate solution 1 mL = 13.81 mg C11H8O2·NaHSO3(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of menadione sodium
bisulfite are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of menadione
sodium bisulfite is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof menadione sodium bisulfite and sodium bisulfite, fillers.

436

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains menadione dimethylpymidinol
bisulfite (C11H8O2·NaHSO3·3H2O) corresponding to 90~130 % of the amount on thelabel.
Confirmation test: According to the amount of this product on the label, the amountcontaining 0.1 g of the raw material for manufacturing of menadione sodium bisulfite isweighed, added with 100 mL of water, shaken up, and filtered. 2 mL of the filtrate isadded with 1 mL of ethanol and 1 mL of ammonia test solution, and shaken up. The
confirmation test ii. for the raw material for manufacturing of menadione sodium bisulfite
is hereinafter applied mutatis mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils and Method
No. 2 is used for the others.
Method No. 1: The amount of this product containing approximately 0.1 g of menadione
sodium bisulfite (C11H8O2·NaHSO3·3H2O) is weighed to three significant digits andthe value is recorded. It is placed in a separatory funnel, added with 30 mL of waterand 30 mL of n-hexane, and vigorously shaken up, and then the aqueous layer isfiltered. The residue on the separatory funnel and the filter paper is washed twice with
10 mL of water each time, and the filtrate and the washings are mixed. The rest of then-hexane layer is added with 20 mL of water and shaken up, and the aqueous layer isfiltered. The residue on the filter paper is washed with 3 mL of water. The filtrate andthe washings are mixed in the previously mixture of the filtrated and washings. The
resulting solution is transferred to a 100 mL volumetric flask, and added with water tothe graduation line to make 100 mL. This is used as a sample solution. Separately,
approximately 0.1 g of menadione sodium bisulfite is weighed to the digits of 0.001 g
and the value is recorded. It is dissolved with water, transferred to a 100 mL
volumetric flask, and added with more water to the graduation line to make 100 mL.
This is used as a standard solution. 5 mL each of the sample solutions is measured into
100 mL brown volumetric flasks T and T', added with 80 mL of water each, mixed
well, added with 2 mL of a mixed solution of ammonia and isopropanol (1:1), andshaken up. The resulting solution in T is, within 30 seconds, added with 4 mL of asolution of ethyl cyanoacetate in isopropanol (3 → 20), shaken up, and then within 30
seconds, added with water to the graduation line to make 100 mL. The resulting
solution in T' is, within 30 seconds, added with water to the graduation line to make100 mL. Separately, 5 mL each of the standard solutions is measured into 100 mL
brown volumetric flasks S and S' using a volumetric pipette, and the same procedure
as for the sample solution is performed. They are allowed to stand for 5 minutes. For

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Provisional Translation from Japanese Original

each solution, absorbances, AT, AT', AS, and AS' at the maximum wavelength around565 nm are measured using water as a control.
Amount of menadione sodium bisulfite (C11H8O2ꞏNaHSO3ꞏ3H2O) (mg)
= Amount of menadione sodium bisulfite for assay (C11H8O2ꞏNaHSO3)
×

AT - AT'× 1.1957
AS - AS'

Method No. 2: The amount of this product containing approximately 0.1 g of menadione
sodium bisulfite (C11H8O2 NaHSO3 3H2O) is weighed to three significant digits andthe value is recorded. It is added with 60 mL of water, vigorously shaken up, andfiltered. The residue on the filter paper is washed twice with 10 mL of water each time.
The filtrate and the washings are mixed, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. This is used as a sample
solution and Method No. 1 is hereinafter applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

(90) Potassium Iodide
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 4-hour drying at 105 °C, it contains
99.0 % or more of potassium iodide (KI).
Physical and chemical properties:
i. This product is colorless or white crystals or white crystalline powder with no odor.
ii. This product is easy to extremely dissolve in water, easy to dissolve in glycerin, andeasy to slightly dissolve in ethanol.
iii. The pH of a solution of this product in water (1 → 10) is 7.0~9.0.
iv. This slightly deliquesces in moist air.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of potassium salt and iodide.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 2 mLof water. The resulting solution shall be colorless and clear.
ii. Heavy metals: 2.0 g (1.95~2.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using

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Provisional Translation from Japanese Original

these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iii. Arsenic: 0.40 g of this product is weighed and a sample solution is prepared by
Method No. 1 of the arsenic test method. The arsenic test is performed by the method
using device A. The color of absorbing solution shall not be darker than the standard
color (5 mg/kg or less).
Loss on drying: Less than 1.0 % (2 g, 105 °C, 4 hours)
Assay: This product is dried at 105 °C for 4 hours, approximately 0.5 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is placed in a stoppered flask, dissolved
with 10 mL of water, added with 35 mL of hydrochloric acid and 5 mL of chloroform,
and titrated with 0.05 mol/L potassium iodate solution while shaking until the reddish
violet color of the chloroform layer disappears. In this case, the end of titration is the time
when the reddish violet color does not appear again within 5 minutes after the chloroform
layer is decolorized.
0.05 mol/L potassium iodate solution 1 mL = 16.60 mg KI(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of potassium iodide
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
iodide is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of potassium
iodide and fillers.
Content: When this product is determined, it contains potassium iodide (KI) corresponding
to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of potassium iodide is weighed,added with 10 mL of water, shaken up well, and filtered. The confirmation test for the
raw materials for manufacturing of potassium iodide is applied to the filtrate mutatis
mutandis.

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Provisional Translation from Japanese Original

Assay: The amount of this product containing approximately 0.5 g of potassium iodide (KI)is weighed to three significant digits and the value is recorded. It is added with 30 mL ofwater, shaken up well, and filtered. The residue on the filter paper is washed twice with
10 mL of water each time. The filtrate and the washings are mixed and Assay for the raw
material for manufacturing of potassium iodide is applied mutatis mutandis.
0.05 mol/L potassium iodide 1 mL = 16.60 mg KI(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
iodide is applied mutatis mutandis.

(91) Folic Acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.0~102.0 % or more of folic acid
(C19H19N7O6) at dehydration product conversion.
Physical and chemical properties:
i. This product is yellow to orange-yellow crystalline powder with no odor.
ii. This product hardly dissolves in water, ethanol, and other organic solvents.
iii. This product dissolves in dilute sodium hydroxide test solution, sodium carbonate
solution (1 → 100), hydrochloric acid, and sulfuric acid. The solution is yellow.
iv. This product gradually changes by light.
Confirmation test:
i. 1.5 mg (1.45~1.54 mg) of this product is dissolved with dilute sodium hydroxide test
solution to make 100 mL. This is used as a sample solution. In the measurement of theabsorption spectrum of this solution, the absorption maximum is at the wavelengths of
255~257 nm, 281~285 nm, and 361~369 nm. When the absorbances at the absorption
maximum of 255~257 nm and 361~369 nm are A1 and A2, respectively, A1/A2 is
2.80~3.00.
ii. When 10 mL of the sample solution in i. is added with a drop of potassium
permanganate test solution, shaken up until the solution turns blue, and observed underultraviolet light within 30 seconds, it emits blue fluorescence.
Purity test:
i. Clarity and color of solution: 0.10 g (0.095~0.104 g) of this product is dissolved with
10 mL of dilute sodium hydroxide test solution. The resulting solution shall be yellow
and clear.

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Provisional Translation from Japanese Original

ii. Free amine: p-aminobenzoyl glutamic acid reference standard is dried in a desiccator(reduced pressure, silica gel) for 4 hours, approximately 0.05 g of it is weighed to thedigits of 0.1 mg, and the value is recorded. It is dissolved with absolute ethanol (2 →5), transferred to a 100 mL volumetric flask, and added with more absolute ethanol (2→ 5) to the graduation line to make 100 mL. Then 3 mL of this solution is measured
using a volumetric pipette, transferred to a 1 L volumetric flask, added with water tothe graduation line to make 1 L. 4 mL of this solution is measured using a volumetricpipette, and the absorbance AS' of this solution is measured in the same procedures as
for S2 solution in the assay. Based on AS' and AC obtained by the assay when theamount of free amine is calculated by the following equation, it shall be 1.0 % or less.
Amount of free amine (%) =

AC W'
×
AS' W

W: Amount of the sample collected by the assay, which is converted into dehydration
product (mg)W′: Amount of p-aminobenzoyl glutamic acid reference standard (mg)Water content: 5 mL of pyridine for Karl Fischer and 20 mL of methanol for Karl Fischer
are put in a dry titration flask, and titrated with Karl Fischer test solution by the end.
Then, approximately 0.2 g of this product is weighed to the digits of 0.001 g and thevalue is recorded. It is placed in a titration flask within 30 seconds, added with a certain
volume of the excess Karl Fischer test solution, stirred for 30 minutes, and tested. The
water content shall be 8.5 % or less.
Ignition residue: Less than 0.5 % (1 g)Assay: Approximately 0.05 each of this product and folic acid reference standard (whosewater content was previously measured in the same manner for this product) is weighedto the digits of 0.1 mg and the value is recorded. Each of them is added with 50 mL of
dilute sodium hydroxide test solution, dissolved by shaking well, transferred to a 100 mL
volumetric flask, added with more dilute sodium hydroxide test solution to the graduationline to make 100 mL, and called T1 and S1 solutions, respectively. 30 mL each of T1 andS1 solutions is measured using a volumetric pipette, transferred to a 100 mL volumetricflask, and added with 20 mL of dilute hydrochloric acid and water to the graduation line
to make 100 mL. 60 mL each is added with 0.5 g (0.45~0.54 g) of zinc powder,repeatedly shaken up, and allowed to stand for 20 minutes. Then, each solution is filteredwith dry filter paper. 10 ml of the first filtrate is removed, and 10 mL of the next filtrate ismeasured using a volumetric pipette, transferred to a 100 mL volumetric flask, and addedwith water to the graduation line to make 100 mL. They are called T2 and S2 solutions,
respectively. 4 mL each of T2 and S2 solutions, is measured using a volumetric pipette,

441

Provisional Translation from Japanese Originaladded with 1 mL of water, 1 mL of dilute hydrochloric acid and 1 mL of sodium nitrite
solution (1 → 1,000), mixed well, allowed to stand for 2 minutes, added with 1 mL ofammonium sulfamate (1 → 200), shaken up well, and allowed to stand for 2 minutes.
Each solution is transferred to a 20 mL volumetric flask, added with 1 mL of N-(1naphthyl)-N'-diethylethylenediamine oxalate (1 → 1,000), shaken up, allowed to standfor 10 minutes, added with water to the graduation line to make 20 mL, and called T3 andS3 solutions, respectively. Separately, 30 mL of the T1 solution is measured using a
volumetric pipette, transferred to a 100 mL volumetric flask, and added with 20 mL of
dilute hydrochloric acid and water to the graduation line to make 100 mL. 4 mL of thissolution is measured using a volumetric pipette and subjected to the same procedure asfor preparing T3 solution from T2 solution. The resulting solution is called C solution.
Separately, 4 mL of water is measured and subjected to the same procedure as for
preparing T3 solution from T2 solution. This is used as a control solution. The
absorbances of AT, AS, and AC at a wavelength 550 nm are measured for T3, S3, and C
solutions. AT' is one-tenth of AC reduced from AT.
Amount of folic acid (C19H19N7O6) (mg)
= Amount of folic acid converted into dehydration product (mg) ×AT'AS(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of folic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of folic acid isapplied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof folic acid and fillers.
Content: When this product is determined, it contains folic acid (C19H19N7O6)
corresponding to 90~115 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1.5 mg of the raw material for manufacturing of folic acid is weighed, addedwith 100 mL of dilute sodium hydroxide test solution for a preparation containing

442

Provisional Translation from Japanese Originalhydrogenated oils, higher saturated fatty acids, fatty acids, vegetable oil or animal fats
added with 30 mL of chloroform, shaken up, and allowed to stand. The aqueous layer isfiltered. The first filtrate of 10 mL is discarded, and the next filtrate is used as a sample
solution. The confirmation tests i. and ii. for the raw material for manufacturing of folicacid are hereinafter applied mutatis mutandis.
Assay: Method No. 1 is used for a preparation containing hydrogenated oils, higher
saturated fatty acids, fatty acids, vegetable oil or animal fats and Method No. 2 is used forthe others.
Method No. 1: The amount of this product containing approximately 0.05 g of folic acid
(C19H19N7O6) is weighed to three significant digits and the value is recorded. It is
added with 50 mL of chloroform, shaken up, and filtered using a glass filter (G4). The
residue on the filter is washed three times with 20 mL of chloroform each time. Whenthe chloroform odor on the residue disappears, the residue is filtered by extracting withapproximately 90 mL of dilute sodium hydroxide test solution and the filtrate is addedwith dilute sodium hydroxide test solution to make 100 mL. This is used as a T1
solution. Separately, approximately 0.05 g of folic acid reference standard (whosewater content was previously measured in the same manner for the raw material for
manufacturing of folic acid) is weighed to the digits of 0.1 mg, and the value isrecorded. It is dissolved with dilute sodium hydroxide test solution, transferred to a
100 mL volumetric flask, and added with more dilute sodium hydroxide test solution
to the graduation line to make 100 mL. This is used as a S1 solution. 30 mL each of T1
and S1 solutions is measured using a volumetric pipette. The assay for the raw material
for manufacturing of folic acid is hereinafter applied mutatis mutandis.
Method No. 2: The amount of this product containing approximately 0.05 g of folic acid
(C19H19N7O6) is weighed to three significant digits and the value is recorded. It is
added with 50 mL of dilute sodium hydroxide test solution, frequently shaken up,
filtered into a 100 mL volumetric flask, and washed with dilute sodium hydroxide test
solution. The filtrate and the washings are mixed and added with more dilute sodiumhydroxide test solution to make 100 mL. This is used as a T1 solution and Method No.
1 is hereinafter applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

443

Provisional Translation from Japanese Original(92) Potassium Iodate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 99.0 % or more of potassium iodate(KIO3).
Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor.
ii. This product is easy to dissolve in water but hard to dissolve in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 7.0~9.0
Confirmation test:
i. When 1 mL of a solution of this product in water (1 → 20) is added with a drop of
starch test solution and 2 drops of hypophosphoric acid test solution (1 → 5), theresulting solution is blue.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions i. ofpotassium salt.
Purity test:
i. Clarity and color of solution: When 1.0 g (0.95~1.04 g) of this product is dissolved
with 20 mL of water, the resulting solution shall be colorless and clear.
ii. Chlorate: When 1.0 g (0.95~1.04 g) of this product is placed in a porcelain dish, addedwith 2 mL of sulfuric acid, and allowed to stand for 10 minutes, chlorine odor or gas
shall not be generated.
iii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is placed in a porcelain pot, addedwith 3 mL of hydrochloric acid (1 → 2), evaporated to dryness on a sand bath, addedwith 3 mL of hydrochloric acid (1 → 2), and again evaporated to dryness on a sandbath. Then, the same procedure is performed three times using 3 mL of hydrochloric
acid each time (1 → 2). The residue is dissolved with 10 mL of water, added with adrop of phenolphthalein test solution, added with ammonia test solution by dropping
until the solution turns slightly red, added with 2 mL of diluted acetic acid, and addedwith water to make 50 mL. This is used as a sample solution. 2.0 mL of lead standard
solution is added with 3 mL of hydrochloric acid (1 → 2), evaporated to dryness on asand bath, and subjected to the same procedure for preparation of the sample solution.
This is used as a control solution. When heavy metal is tested using these solutions, thecolor of the sample solution shall not be darker than that of the control solution (10mg/kg and less).
iv. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the arsenic test method. The arsenic test is performed by444

Provisional Translation from Japanese Original

the method using device A. The color of absorbing solution shall not be darker than
the standard color (5 mg/kg or less).
Assay: Approximately 1.0 g of this product is weighed to the digits of 0.01 g and the value
is recorded. It is dissolved with 50 mL of water, transferred to a 100 mL volumetric flask,
and added with water to the graduation line to make 100 mL. 10 mL of this solution ismeasured using a volumetric pipette, transferred to a stoppered flask, added with 30 mL
of water, 3 g (2.5~3.4 g) of potassium iodide and 5 mL of sulfuric acid (1 → 5), tightly
stoppered within 30 seconds, allowed to stand in a dark place for 5 minutes, and titrated
with 0.1 mol/L sodium thiosulfate solution (indicator: 1 mL of starch test solution). A
blank test is performed in the same manner and corrections are made.
0.1 mol/L sodium thiosulfate solution 1 mL = 3.567 mg KIO3(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of potassium iodate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
iodate is applied mutatis mutandis.

(93) Calcium Iodate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 95.0 % or more of calcium iodate[Ca(IO3)2].
Physical and chemical properties:
i. This product is white crystals or crystalline powder with no odor or a slightly specificodor.
ii. This product is hard to dissolve in water and ethanol.
Confirmation test:
i. When 5 mL of a saturated solution of this product in water is added with a drop of
starch test solution and 2 drops of hypophosphoric acid test solution (1 → 5), theresulting solution is blue.
ii. Flame coloration test for this product moisturized with hydrochloric gives red color.

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Provisional Translation from Japanese Original

Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is added with 50 mLof 1 mol/L hydrochloric acid test solution, and dissolved on a water bath by warming.
The resulting solution shall be colorless to pale yellow and clear.
ii. Chlorate: The purity test ii. for the raw material for manufacturing of potassium iodate
is applied mutatis mutandis.
iii. Heavy metal: The purity test iii. for the raw material for manufacturing of potassium
iodate is applied mutatis mutandis (10 mg/kg or less).
iv. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed, added with 5 mL ofchlorine (2 → 3), evaporated to dryness on a water bath, added with 5 ml ofhydrochloric acid (2 → 3), and again evaporated to dryness on a water bath. This
procedure is repeated until the color of iodine disappears, and then it is added with 5
mL of water, and dissolved by heating on a water bath to prepare a sample solution.
Arsenic is tested by the method using device A, the color of this sample solution shallnot be darker than that obtained when the same procedure is performed without using
this product, and 2.0 mL of arsenic standard solution is added and thereinafter thesame procedure as for the sample solution is performed (5 mg/kg or less).
Assay: 0.1 g of this product is weighed to the digits of 0.001 g and the value is recorded. It
is dissolved with 30 mL of water, 3 g (2.5~3.4 g) of potassium iodide, and 5 mL ofhydrochloric acid (1 → 9), and titrated with 0.1 mol/L sodium thiosulfate solution(indicator: 1 mL of starch test solution). A blank test is performed in the same mannerand corrections are made.
0.1 mol/L sodium thiosulfate solution 1 mL = 3.249 mg Ca (IO3)2(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium iodate areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium iodate
is applied mutatis mutandis.

446

Provisional Translation from Japanese Original(94) Riboflavin
Riboflavin (part 1)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 2-hour drying at 105 °C, it contains
96.0 % or more of riboflavin (C17H20N4O6).
Physical and chemical properties:
i. This product is yellow to orange-yellow crystals with a slightly specific odor.
ii. This product is hard to extremely dissolve in water and hardly dissolves in ethanol,
ether, or chloroform.
iii. This product dissolves in 1 mol/L sodium hydroxide test solution.
iv. A saturated solution of this product is neutral.
v. This product decomposes by light.
vi. Melting point: Approximately 280 °C (dissolution).
Confirmation test:
i. A solution of this product in water (1 → 100,000) is pale yellow-green and emits
strong yellow-green fluorescence. When 5 mL of this product is added with 0.02 g(0.015~0.024 g) of hydrosulfite sodium, the color of the solution and the fluorescence
disappear, but when it is shaken up in air they gradually reappear. The fluorescence ofthe solution disappears when dilute hydrochloric acid or 1 mol/L sodium hydroxide
test solution is added by dropping.
ii. When 10 mL of a solution of this product in water (1 → 100,000) is put in a stoppered
test tube, added with 1 mL of 1 mol/L sodium hydroxide test solution, irradiated with
10~30 watts of fluorescent light at a distance of 20 cm at 20~40 °C for 30 minutes,
added with 0.5 mL of acetic acid to make it acidic, added with 5 mL of chloroform,and shaken up well, the chloroform layer emits yellow-green fluorescence.
iii. In the measurement of the absorption spectrum of a solution of this product in pH 7.0
phosphate buffer (1 → 100,000), the absorption maximum is at the wavelengths of
265~267 nm, 372~374 nm, and 444~446 nm. When the absorbances are called A1, A2,
and A3, respectively, A2/A1 and A3/A1 are 0.314~0.333 and 0.364~0.388, respectively.
Purity test:i. Specific rotation: This product is dried at 105 °C for 2 hours, approximately 0.1 g of it
is weighed to the digits of 0.001 g, and the value is recorded. It is added with 4 mL of
dilute sodium hydroxide test solution using a volumetric pipette to dissolve, addedwith 10 mL of newly boiled and cooled water, added with 4 mL of non-aldehyde
ethanol using a volumetric pipette while shaking well, transferred to a 20 mL

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Provisional Translation from Japanese Original

volumetric flask, and added with newly boiled and cooled water to the graduation line
to make 20 mL. When rotation of this solution is measured within 30 minutes, it shallbe [α]20
D = -120 ~ -140°.

ii. Lumiflavin: 0.025 g (0.0245~0.0254 g) of this product is added with 10 mL ofchloroform without ethanol, shaken up for 5 minutes, and filtered. The color of thisfiltrate shall not be darker than that of a solution in which 4.0 mL of 0.017 mol/L
potassium dichromate is added with water to make 1 L.
Loss on drying: Less than 1.5 % (0.5 g, 105 °C, 2 hours)
Ignition residue: Less than 0.30 % (1 g)Assay: The procedure is performed using a lightproof container away from direct sunlight.
This product is dried at 105 °C for 2 hours, approximately 0.015 g of it is weighed to thedigits of 0.1 mg, and the value is recorded. It is added with 800 mL of glacial acetic acid
(1 → 400), dissolved by warming, allowed to stand, transferred to a 1 L volumetric flask,
and added with water to the graduation line to make 1 L. This is used as a sample
solution. Separately, riboflavin reference standard is dried at 105 °C for 2 hours,
approximately 0.015 of it is weighed to the digits of 0.1 mg, and the value is recorded. It
is transferred to a 1 L volumetric flask, added with 800 mL of glacial acetic acid (1 →400), dissolved by warming, allowed to stand, and added with water to the graduationline to 1 L. This is used as a standard solution. The absorbances of AT and AS of thesample and standard solutions, respectively, at a wavelength of 445 nm are measured
using water as a control solution. Then, 5 mL each of these solutions is added with 0.02 g(0.015~0.024 g) of sodium hydrosulfite and shaken up to be decolorized, and theabsorbances of AT' and AS' of these solutions are measured within 30 seconds.
Amount of riboflavin (C17H20N4O6) (mg)
= Amount of riboflavin reference standard (mg) ×(b) Standard of storage method

AT - AT'AS - AS'

It shall be stored in a lightproof airtight container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of riboflavin (part 1)
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of riboflavin (part1) is applied mutatis mutandis.

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Provisional Translation from Japanese Original

C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof riboflavin (part 1) and fillers.
Content: When this product is determined, it contains riboflavin (C17H20N4O6)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 mg of
the raw material for manufacturing of riboflavin (part 1) is weighed, added with 100
mL of water, shaken up, and filtered. To the filtrate, the confirmation test i. for the raw
material for manufacturing of riboflavin (part 1) is hereinafter applied mutatis
mutandis.
ii. 10 mL of the filtrate in i. is put in a stoppered test tube, the confirmation test ii. for the
raw material for manufacturing of riboflavin (part 1) is hereinafter applied mutatis
mutandis.
Assay: The procedure is performed using a lightproof container away from direct sunlight.
The amount of this product containing approximately 0.015 g of riboflavin (C17H20N4O6)
is weighed to three significant digits and the value is recorded. It is added with 800 mL ofglacial acetic acid (1 → 400), dissolved by warming, allowed to stand, transferred to a 1
L volumetric flask, added with water to the graduation line to make 1 L, and filtered withdry filter paper. This is used as a sample solution. Separately, riboflavin reference
standard is dried at 105 °C for 2 hours, and the assay for the raw material for
manufacturing of riboflavin (part 1) is hereinafter applied mutatis mutandis.
Amount of riboflavin (C17H20N4O6) (mg)
= Amount of riboflavin reference standard (mg) ×

AT - AT'AS - AS'(b) Standard of storage method
It shall be stored in a lightproof capped container.
Riboflavin (part 2)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 3-hour drying at 105 °C, it contains
80.0 % or more of riboflavin (C17H20N4O6).
Physical and chemical properties:
i. This product is orange-yellow to yellow-brown fine particles with a specific odor.

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Provisional Translation from Japanese Original

ii. This product is hard to extremely dissolve in water and hardly dissolves in ethanol,
ether, or chloroform.
iii. This product decomposes by light.
Confirmation test:
i. The confirmation test i. for the raw material for manufacturing of riboflavin (part 1) isapplied mutatis mutandis.
ii. The confirmation test ii. for the raw material for manufacturing of riboflavin (part 1) isapplied mutatis mutandis.
iii. In the measurement of the absorption spectrum of a solution of this product in pH 7.0
phosphate buffer (1 → 100,000), the absorption maximum is at the wavelengths of
264~268 nm, 371~375 nm, and 443~447 nm. When the absorbances are called A1, A2,
and A3, respectively, A2/A1 and A3/A1 are 0.310~0.333 and 0.364~0.388, respectively.
Purity test:i. Specific rotation: This product is dried at 105 °C for 3 hours, approximately 0.1 g of it
is weighed to the digits of 0.001 g, and the value is recorded. It is added with 4 mL of
dilute sodium hydroxide test solution using a volumetric pipette to dissolve, addedwith 10 mL of newly boiled and cooled water, added with 4 mL of non-aldehyde
ethanol using a volumetric pipette while shaking well, transferred to a 20 mL
volumetric flask, added with newly boiled and cooled water to the graduation line to
make 20 mL, and centrifuged. When rotation of the supernatant liquid is measured
within 30 minutes, it shall be [α]20
D = -100 ~ -120°.

ii. Lumiflavin: The purity test ii. for the raw material for manufacturing of riboflavin(part 1) is applied mutatis mutandis.
iii. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: Less than 5.0 % (1 g, 105 °C, 3 hours)
Ignition residue: Less than 3.0 % (1 g)Assay: The procedure is performed using a lightproof container away from direct sunlight.
This product is dried at 105 °C for 3 hours, approximately 0.05 g of it is weighed to the450

Provisional Translation from Japanese Original

digits of 0.1 mg, and the value is recorded. It is added with 500 mL of 10 w/v% saccharin
sodium solution, stirred for 30 minutes, and centrifuges as appropriate. This is used as asample stock solution. 10 mL of the sample stock solution is measured using a volumetricpipette, transferred to a 100 mL volumetric flask, added with water to the graduation line
to make 100 mL, and filtered with a 0.45 µm membrane filter. This is used as a sample
solution. 20 µL of this solution is tested by the liquid chromatography under thefollowing conditions. The peak height or area of riboflavin is measured from the obtained
chromatogram, the concentration of riboflavin is determined from a calibration curve
separately obtained, and the content is calculated.
Operating conditions:
Detector: Fluorescence detector (excitation wavelength: 445 nm, fluorescencewavelength: 530 nm)
Column: A stainless tube (inner diameter: approx. 4.0 mm, length: approx. 300 mm) isfilled with 10 µm or less of octadecyl-silylated silica gel.
Column temperature: 40 °C
Mobile phase: Water / methanol (7:3)
Flow rate: 0.8 mL/min.
Preparation of calibration curve:Riboflavin reference standard is dried at 105 °C for 3
hours, approximately 25 mg of it is weighed to the digits of 0.1 mg, and the value isrecorded. It is dissolved with 10 w/v% saccharin sodium solution, transferred to a 250
mL brown stoppered Erlenmeyer flask, and added with the same solution to thegraduation line to prepare a standard stock solution of riboflavin (1 mL of this solution
contains 0.1 mg of riboflavin (C17H20N4O6)). In the use, a certain volume of the standard
stock solution is accurately diluted with water to prepare the solutions containing 5, 7.5,
and 10 µg/mL. Each solution is filtered with a 0.45 µm membrane filter to prepare astandard solution. 20 µL each of the standard solutions is hereinafter tested by the liquid
chromatography as is the case in the sample solution. The peak height or area ofriboflavin is measured from the obtained chromatogram and a calibration curve isprepared.
(b) Standard of manufacturing method
For manufacturing, the riboflavin producing strain of Ashbya gossypii is aerobicallycultured.After the cultivation, the bacterial cells are heat-treated to separate riboflavin, anddried.
(c) Standard of storage method
It shall be stored in a lightproof airtight container.

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Provisional Translation from Japanese Original(d) Standard of the label
The following words shall be described on the immediate container or the immediatewrapper of this product.
Riboflavin (Feed grade)
B. Preparation(a) Compositional standards
The compositional standard of the raw material for manufacturing of riboflavin (part 2) isapplied mutatis mutandis.
(b) Standard of manufacturing method
The standard of manufacturing method of the raw material for manufacturing of
riboflavin (part 2) is applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of riboflavin (part2) is applied mutatis mutandis.
(d) Standard of the label
The standard of the label of the raw material for manufacturing of riboflavin (part 2) isapplied mutatis mutandis.
Riboflavin (part 3)
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 2-hour drying at 105 °C, it contains
96.0 % or more of riboflavin (C17H20N4O6).
Physical and chemical properties:
i. This product is yellow to orange-yellow crystals with a slightly specific odor.
ii. This product is hard to extremely dissolve in water and hardly dissolves in ethanol,
ether, or chloroform.
iii. This product dissolves in 1 mol/L sodium hydroxide test solution.
iv. A saturated solution of this product is neutral.
v. This product decomposes by light.
vi. Melting point: Approximately 290 °C (dissolution)
Confirmation test: The confirmation test of the raw material for manufacturing of riboflavin(part 1) is applied mutatis mutandis.
Purity test: The purity test of the raw material for manufacturing of riboflavin (part 1) isapplied mutatis mutandis.
Loss on drying: Less than 1.5 % (0.5 g, 105 °C, 2 hours)

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Provisional Translation from Japanese Original

Ignition residue: Less than 0.30 % (1 g)Assay: The assay of the raw material for manufacturing of riboflavin (part 1) is appliedmutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the riboflavin producing recombinant, whose host is the strain
belonging to Bacillus subtilis, is aerobically cultured. After the cultivation, the bacterial
cells are heat-treated to separate crude crystal fractions of riboflavin. Then the crude crystals
are purified and the obtained solid is dried.
(c) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation (part 1)(a) Compositional standards
The compositional standard of the raw material for manufacturing of riboflavin (part 3) isapplied mutatis mutandis.
(b) Standard of manufacturing method
The standard of manufacturing method of the raw material for manufacturing of
riboflavin (part 3) is applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of riboflavin (part3) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof riboflavin (part 3) and fillers.
Content: When this product is determined, it contains riboflavin (C17H20N4O6)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 mg of
the raw material for manufacturing of riboflavin (part 3) is weighed, added with 100
mL of water, shaken up, and filtered. To the filtrate, the confirmation test i. for the raw
material for manufacturing of riboflavin (part 1) is applied mutatis mutandis.
ii. 10 mL of the filtrate in i. is put in a stoppered test tube, and the confirmation test ii. forthe raw material for manufacturing of riboflavin (part 1) is applied mutatis mutandis.
Assay: The procedure is performed using a lightproof container away from direct sunlight.
The amount of this product containing approximately 0.015 g of riboflavin (C17H20N4O6)
is weighed to three significant digits and the value is recorded. It is added with 800 mL of453

Provisional Translation from Japanese Original

glacial acetic acid (1 → 400), dissolved by warming, allowed to cool, transferred to a 1 L
volumetric flask, added with water to the graduation line to make 1 L, and filtered withdry filter paper. This is used as a sample solution. Separately, riboflavin reference
standard is dried at 105 °C for 2 hours, and the assay for the raw material for
manufacturing of riboflavin (part 1) is hereinafter applied mutatis mutandis.
Amount of riboflavin (C17H20N4O6) (mg)
= Amount of riboflavin reference standard (mg) ×

AT - AT'AS - AS'(b) Standard of storage method
It shall be stored in a lightproof capped container.

(95) Riboflavin Butyrate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried in a desiccator (reduced pressure, silica gel) for 4 hours
and determined, it contains 97.0~102.0 % of riboflavin butyrate (C33H44N4O10).
Physical and chemical properties:
i. This product is yellow-orange crystals or crystalline powder with a slight specific odor.
ii. This product is easy to extremely dissolve in ethanol and chloroform and hardly
dissolves in water.
iii. Melting point: Approximately 148 °C.
Confirmation test:
i. A solution of this product in ethanol (1 → 100,000) is pale yellow-green and emits
strong yellow-green fluorescence. This fluorescence disappears when dilutehydrochloric acid or 1 mol/L sodium hydroxide test solution is added.
ii. 0.01 g (0.005~0.014 g) of this product is weighed, dissolved with 5 mL of ethanol,
added with 2 mL of a mixed solution of equivalent volume of hydroxylaminehydrochloride solution (3 → 20) and sodium hydroxide solution (3 → 20), shaken up
well, and added with 0.8 mL of hydrochloric acid, 0.5 mL of ferric chloride test
solution, and 5 mL of ethanol. The resulting solution is dark red-brown.
Purity test:
i. Clarity and color of solution: 0.10 g (0.095~0.104 g) of this product is dissolved with
10 mL of chloroform. The resulting solution shall be clear.
ii. Ratio of absorbance: In the measurement of the absorption spectrum of a solution ofthis solution in ethanol (1.7 → 100,000), the absorption maximum is at the454

Provisional Translation from Japanese Original

wavelengths of 269~271 nm, 349~351 nm, and 444~446 nm. When the absorbances at
the absorption maximums are called A1, A2, and A3, respectively, A1/A2, A1/A3, and,A2/A3 shall be 3.50~3.90, 2.47~2.77, and 0.65~0.75, respectively.
Loss on drying: Less than 1.0 % (1 g, reduced pressure, silica gel, 4 hours)
Ignition residue: Less than 0.5 % (1 g)Assay: This product is dried for 4 hours in a desiccator (reduced pressure, silica gel),approximately 0.04 g of it is weighed to the digits of 0.1 mg, and the value is recorded. It
is dissolved with ethanol, transferred to a 500 mL volumetric flask, and added withethanol to the graduation line to make 500 mL. 10 mL of this solution is measured using a
volumetric pipette, transferred to a 50 mL volumetric flask, and added with ethanol to thegraduation line to make 50 mL. This is used as a sample solution. Separately, riboflavinreference standard is dried at 105 °C for 3 hours, approximately 0.05 g of it is weighed tothe digits of 0.1 mg, and the value is recorded. It is added with 4 mL of glacial acetic acidand 150 mL of water, dissolved by warming, allowed to cool, transferred to a 500 mL
volumetric flask, and is added with water to the graduation line to make 500 mL. 5 mL ofthis solution is measured using a volumetric pipette, transferred to a 50 mL volumetricflask, and added with ethanol to the graduation line to make 50 mL. This is used as astandard solution. The absorbances of AT and AS of the sample and standard solutions at awavelength of 445 nm are measured, using ethanol as a control solution.
Amount of riboflavin butyrate (C33H44N4O10) (mg)
= Amount of riboflavin reference standard (mg) ×AT× 0.8725
AS(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of riboflavin butyrate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of riboflavinbutyrate is applied mutatis mutandis.

(96) Zinc Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards

455

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains 80.0 % or more of zinc sulfate (dry)(ZnSO4).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product is easy to dissolve in water, easy to slightly dissolve in glycerin, andhardly dissolves in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 4.5~6.5
iv. This product is hygroscopic.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of zinc salt and sulfate.
Purity test:
i. Clarity and color of solution: 0.25 g (0.245~0.254 g) of this product is dissolved with 5
mL of water and 1 mL of sulfuric acid (1 → 20). The resulting solution shall be clear.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is placed in a Nessler tube, dissolved
with 10 mL of water, added with 20 mL of potassium cyanide test solution, shaken up
well, added with 2 drops of sodium sulfide test solution, and after 5 minutes isobserved from above using a white paper background. Then, the color of this solution
shall not be darker than that of a solution prepared by the procedure in which 2.0 mLof lead standard solution is added with 8.0 mL of water and 20 mL of potassium
cyanide test solution, shaken up well, and hereinafter subjected to the same procedures
as for the sample solution (20 mg/kg or less).
iii. Arsenic: 0.20 g (0.195~0.204 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (10 mg/kg or less).
Assay: Approximately 0.2 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with water, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. 25 mL of this solution ismeasured using a volumetric pipette, added with 100 mL of water and 2 mL of pH 10.7
ammonia-ammonium chloride buffer and titrated with 0.01 mol/L ethylene-diaminetetraacetic acid disodium solution (indicator: 0.04 g (0.035~0.044 g) of eriochrome black
T-sodium chloride indicator).
0.01 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL
=1.614 mg ZnSO4(b) Standard of storage method
It shall be stored in a capped container.

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Provisional Translation from Japanese Original

B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of zinc sulfate (dry)are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc sulfate(dry) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of zinc
sulfate (dry) and fillers.
Content: When this product is determined, it contains zinc sulfate (ZnSO4) corresponding to
90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of zinc sulfate (dry) is weighed,added with 10 mL of water, shaken up well, and filtered. To the filtrate the confirmation
test for the raw material for manufacturing of zinc sulfate (dry) is hereinafter appliedmutatis mutandis.
Assay: The amount of this product containing approximately 0.2 g of zinc sulfate (ZnSO4)
is weighed to three significant digits and the value is recorded. The assay for the raw
material for manufacturing of zinc sulfate (dry) is hereinafter applied mutatis mutandis.
0.01 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL
= 1.614 mg ZnSO4(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc sulfate(dry) is applied mutatis mutandis.

(97) Zinc Sulfate (crystal)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 99.0~102.0 % of zinc sulfate(ZnSO4 7H2O).
Physical and chemical properties:
i. This product is colorless crystals or white crystalline powder with no odor.

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Provisional Translation from Japanese Original

ii. This product is easy to extremely dissolve in water, easy to dissolve in glycerin, andhardly dissolves in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 3.5~6.0.
iv. This product effloresces in dry air.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of zinc salt and sulfate.
Purity test:
i. Clarity and color of solution: 0.25 g (0.245~0.254 g) of this product is dissolved with 5
mL of water. The resulting solution shall be clear.
ii. Heavy metal: The purity test ii. for the raw material for manufacturing of zinc sulfate(dry) is applied mutatis mutandis. In this case, “2.0 mL” and “8.0 mL” shall be
replaced by “1.0 mL” and “9.0 mL” respectively (10 mg/kg or less).
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (5 mg/kg or less).
Assay: Approximately 0.4 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with water, transferred to a 100 mL volumetric flask, andadded with water to the graduation line to make 100 mL. 25 mL of this solution ismeasured using a volumetric pipette, added with 100 mL of water and 2 mL of pH 10.7
ammonia-ammonium chloride buffer and titrated with 0.01 mol/L ethylene-diaminetetraacetic acid disodium solution (indicator: 0.04 g (0.035~0.044 g) of eriochrome black
T-sodium chloride indicator).
0.01 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL
= 2.876 mg ZnSO4 7H2O(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of zinc sulfate(crystal) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc sulfate(crystal) is applied mutatis mutandis.

458

Provisional Translation from Japanese Original(98) Zinc Sulfate Methionine
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 2-hour drying at 110 °C, it contains
20.0~23.5 % of zinc (Zn) and 38.0~44.0 % of methionine (C5H11NO2S).
Physical and chemical properties:
i. This product is pale purple powder with a specific odor.
ii. This product is hard to dissolve in water and hardly dissolves in ethanol and .methanoliii. The pH of a solution of this product in water (1 → 100) is 4.2~4.7.
Confirmation test:
i. When 10 mL of a solution of this product in dilute hydrochloric acid (1 → 10) is addedwith several drops of ammonium sulfide test solution or sodium sulfide test solution,
gray-brown precipitation is generated. The precipitation is fractionated. It does notdissolve by adding diluted acetic acid but dissolves by adding dilute hydrochloric acid.
ii. When 10 mL of a solution of this product in dilute hydrochloric acid (1 → 10) is
added with several drops of potassium ferrocyanide test solution, blue-white
precipitation is generated. It does not dissolve when dilute hydrochloric acid is added
to a part of it, but with the addition of 1 mol/L sodium hydroxide test solution, anotherpart of it dissolves.
iii. A solution of this product in water (1 → 100) gives the qualitative reaction i. of
sulfate.
iv. 0.5 g (0.45~0.54 g) of this product and 0.1 g (0.05~0.14 g) of the raw material for
manufacturing of DL-methionine are weighed. Each of them is dissolved with 10 mLof water to prepare sample and standard solutions. 5 µL each of the sample andstandard solutions is spotted on a thin layer plate prepared using silica gel for thinlayer chromatography. Then it is developed approximately 10~15 cm with thedeveloping solvent, a mixed solution of ethanol and water (63:37), and the thin layer isdried at 105 °C for 30 minutes. When it is evenly sprayed with a solution of ninhydrin
in ethanol (1 → 200) and heated at 105 °C for 5 minutes, the spots obtained from thesample and standard solutions are purple, and their Rf values are equal.
v. 1 mg (0.5~1.4 mg) of this product is weighed. When the infrared absorption spectrumof this product is measured using the potassium bromide disk method of the infraredabsorption spectroscopy, absorptions are observed at around 1,640 cm−1, 1,490 cm−1,
1,420 cm−1, 1,340 cm−1, 1,120 cm−1, 1,060 cm−1, 980 cm−1 and 610 cm−1, but notobserved at around 2,100 cm−1.

459

Provisional Translation from Japanese Original

Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and ashed by ignition at
500~600 °C. After cooling, it is added with 5 mL of hydrochloric acid (1 → 2) andevaporated to dryness on a water bath. The residue is added with 15 mL of water and 2
drops of hydrochloric acid (1 → 2) and heated on a water bath for 30 minutes. Then, it
is added with a drop of phenolphthalein test solution, added with ammonia test
solution by dropping until the solution turns slightly red, and added with 2 mL ofacetic acid. It is added with 30 mL of potassium cyanide test solution, stirred, andfiltered. The filtrate and the washings are transferred to a Nessler tube and added withwater to make 50 mL. When it is added with 2 drops of sodium sulfide test solution
and allowed to stand for 5 minutes, the color of this solution shall not be darker thanthat of a solution prepared by the procedure in which 3.0 mL of lead standard solution
is added with 15 mL of water and 30 mL of potassium cyanide test solution, stirred
well, added with 2 drops of sodium sulfide test solution and allowed to stand for 5
minutes (30 mg/kg or less).
ii. Arsenic: 0.67 g (0.665~0.674 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (3 mg/kg or less).
Loss on drying: Less than 15.0 % (1 g, 110 °C, 2 hours)
Assay
i. Zinc: This product is dried at 110 °C for 2 hours, approximately 0.2 g of it is weighedto the digits of 0.001 g, and the value is recorded. It is placed in a 100 mL volumetricflask, dissolved with water, and added with more water to the graduation line to make100 mL. 25 ml of this solution is measured using a volumetric pipette, added with 100
mL of water and 2 mL of pH 10.7 ammonia-ammonium chloride buffer and titrated
with 0.05 mol/L ethylene-diamine-tetraacetic acid disodium solution (indicator: 0.04 g(0.035~0.044 g) of eriochrome black T-sodium chloride indicator).
0.05 mol/L ethylene-diamine-tetraacetic acid disodium solution 1 mL = 3.269 mg
Zn
ii. Methionine: This product is dried at 110 °C for 2 hours, approximately 0.6 g of it isweighed to the digits of 0.001 g, and the value is recorded. The assay for the raw
material for manufacturing of DL-methionine is hereinafter applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a capped container.

460

Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of zincmethionine sulfate and fillers.
Content: When this product is determined, it contains zinc (Zn) and methionine (C5H11NO
2S) corresponding to 90~110 % of the amount on the label.

Confirmation test:i. According to the amount of this product on the label, the amount containing 0.4 g ofzinc (Zn) is weighed, added with 10 mL of dilute hydrochloric acid, shaken up well,and filtered. When the filtrate is added with several drops of ammonium sulfide or
sodium sulfide test solution, gray precipitation is generated. The fractionatedprecipitation does not dissolve by adding diluted acetic acid but dissolves by adding
dilute hydrochloric acid.
ii. According to the amount of this product on the label, the amount containing 0.4 g ofzinc (Zn) is weighed, added with 10 mL of dilute hydrochloric acid, shaken up well,and filtered. When the filtrate is added with several drops of potassium ferrocyanide
test solution, pale yellow-white precipitation is generated. The precipitation does notdissolve when dilute hydrochloric acid is added to a part of it, but with the addition of
1 mol/L sodium hydroxide test solution, another part of it dissolves.
iii. According to the amount of this product on the label, the amount containing 0.4 g ofzinc (Zn) is weighed, added with 100 mL of water, stirred, and filtered. The filtrate
gives the qualitative reaction i. of sulfate.
iv. According to the amount of this product on the label, the amount containing 0.1 g ofmethionine (C5H11NO2S) is weighed. Separately, 0.1 g (0.05~0.14 g) of the raw
material for manufacturing of methionine is weighed. Each of them is dissolved with
10 mL of water and filtered. Both filtrates are used as sample and standard solutions. 5µL each of the sample and standard solutions is spotted on a thin layer plate preparedusing silica gel for thin-layer chromatography. Then it is developed approximately
10~15 cm with the developing solvent, a mixed solution of ethanol and water (63:37),
and the thin layer is dried at 105 °C for approximately 30 minutes. When it is evenly
sprayed with a solution of ninhydrin in ethanol (1 → 200) and heated at 105 °C for 5
minutes, the spots obtained from the sample and standard solutions are purple, andtheir Rf values are equal.
v. According to the amount of this product on the label, the amount containing 1 mg of
the raw material for manufacturing of zinc sulfate methionine. When the infraredabsorption spectrum of it is measured using the potassium bromide disk method of the461

Provisional Translation from Japanese Original

infrared absorption spectroscopy, absorptions are observed at around 1,640 cm-1, but
not observed at around 2,100 cm−1.
Assay:
i. Zinc: The amount of this product containing approximately 0.02 g of zinc sulfate
methionine is weighed to three significant digits and the value is recorded. It is addedwith hydrochloric acid (1 → 11), stirred for 30 minutes, filtered, transferred to a 100mL volumetric flask, and added with water to the graduation line to make 100 mL. 5
mL of the filtrate is measured using a volumetric pipette, and added with 100 mL ofhydrochloric acid (1 → 100) to make 100 mL. This is used as a sample solution.
Separately, the same procedure is performed without adding this product to prepare ablank test solution. Then 10, 20, 30, 40 and 50 mL of lead standard solution aremeasured by a volumetric pipette and added with hydrochloric acid (1 → 100) to make100 mL, and they are called the standard solutions 1, 2, 3, 4 and 5. The sample
solution, the standard solutions 1, 2, 3, 4 and 5, and the blank test solution aredetermined by the atomic absorption spectrophotometry. A calibration curve isprepared from the absorbances of the standard solutions 1, 2, 3, 4 and 5, and thecontent of zinc in the sample is calculated.
Gas to be used: Flammable gas, acetylene
Combustion-supporting gas, air
Lamp: Zinc hollow cathode lamp
Wavelength: 213.9 nm
Content of zinc (%)=

Concentration of zinc obtained from the calibration curve (µg/mL) × 200
Collected amount of the sample (mg)ii. Methionine: The amount of this product containing approximately 0.03 g ofmethionine (C5H11NO2S) is weighed to three significant digits and the value isrecorded. It is added with 15 mL of a mixed solution of n-hexane and isopropanol(1:1), stirred to dissolve, added with 70 mL of hydrochloric acid (9 → 250), vigorously
stirred and extracted. This solution is transferred to a separatory funnel while filteringwith absorbent cotton and the container is washed with 5 mL of a mixed solution of nhexane and isopropanol (1:1) and 15 mL of hydrochloric acid (9 → 250). The
washings are poured into the absorbent cotton and these filtrates are mixed. The
resulting solution is vigorously shaken up and the aqueous layer is transferred to a 100mL volumetric flask and added with hydrochloric acid (9 → 250) to the graduationline to make 100 mL. 2 mL of this solution is measured using a volumetric pipette,added with 2 mL of hydrochloric acid (9 → 250) and 12 mL of water using a462

Provisional Translation from Japanese Original

volumetric pipette. This is used as a sample solution. Separately, 0.030 g of dry DLmethionine for assay is weighed to the digits of 0.1 mg and the value is recorded. It isdissolved with hydrochloric acid (9 → 250), transferred to a 100 mL volumetric flask,
added with hydrochloric acid (9 → 250) to the graduation line to make 100 mL. 2 mLof this solution is measured using a volumetric pipette, added with 2 mL ofhydrochloric acid (9 → 250) and 12 mL of water using a volumetric pipette. This isused as a standard solution. Separately, 2 mL of hydrochloric acid (9 → 250) is
measured using a volumetric pipette, added with 6 mL of water using a volumetricpipette to prepare a blank test solution. Then, 2 mL each of the sample, standard, andblank test solutions is measured using a volumetric pipette, transferred to a 25 mL
volumetric flask, added with 1 mL of newly prepared ninhydrin-ascorbic acid test
solution and 1 mL of pyridine using a volumetric pipette, shaken up, heated in a water
bath for 25 minutes, cooled with water, and added with water to the graduation line to
make 25 mL. The blank test solution is used as a control solution, and the absorbancesof AT and AS of sample and standard solutions at a wavelength 570 nm are measured.
Amount of methionine (C5H11NO2S) (mg)
= Amount of DL-methionine for assay (mg) ×ATAS(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc sulfate
methionine is applied mutatis mutandis.

(99) Cobalt Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 87.0 % or more of cobalt sulfate(CoSO4).
Physical and chemical properties:
i. This product is pale pink powder with no odor.
ii. This product gradually dissolves in water and is hard to slightly dissolve in ethanol.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 200) is added with 0.5 mL of 1
mol/L sodium hydroxide test solution, blue precipitation is generated. It changes tobrown by heating.
ii. A solution of this product in water (1 → 20) gives the qualitative reaction of sulfate.

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Provisional Translation from Japanese Original

Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of dilute hydrochloric acid. The resulting solution shall be red and clear.
ii. Lead: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with nitric acid (1 →150), transferred to a 20 mL volumetric flask, and added with more nitric acid (1 →150) to the graduation line to make 20 mL. When lead is tested, using this solution as asample solution, by the lead test method (Method No. 1 of the atomic absorption
spectrophotometry), the amount of lead shall be 20 mg/kg or less.
iii. Arsenic: 2.0 g (1.95~2.04 g) of this product is weighed, dissolved with 50 mL ofwater and 5 mL of dilute hydrochloric acid, transferred to a 100 mL volumetric flask,
and added with water to the graduation line to make 100 mL. 10 mL of this solution ismeasured using a volumetric pipette to prepare a sample solution. When arsenic istested using the sample solution by the method using device B, the color of mercuricbromide paper shall not be darker than the standard color (10 mg/kg or less).
Assay: Appropriately 0.4 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is transferred to a 100 mL volumetric flask, dissolved with 5 mL of dilutehydrochloric acid and 20 mL of water, and added with more water to the graduation line
to make 100 mL. 10 mL of this product is measured using a volumetric pipette, addedwith 100 mL of water, and titrated with 0.01 mol/L ethylenediaminetetraacetic aciddisodium solution, while adjusting to pH 8.0 with ammonia test solution (indicator: 0.2 g(0.15~0.24 g) of murexide indicator).
0.01 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL
= 1.550 mg CoSO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of cobalt sulfate (dry)are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of cobalt sulfate(dry) is applied mutatis mutandis.

464

Provisional Translation from Japanese Original(100) Cobalt Sulfate (crystal)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.0~103.0 % of cobalt sulfate(CoSO4 7H2O).
Physical and chemical properties:
i. This product is glossy, dark red, transparent crystals or pink-white sandy crystals withno odor.
ii. This product is easy to dissolve in water and hardly dissolves in ethanol.
Confirmation test:
i. When 5 mL of a solution of this product in water (1 → 100) is added with 0.5 mL of 1
mol/L sodium hydroxide test solution, blue precipitation is generated. It changes tobrown by heating.
ii. A solution of this product in water (1 → 10) gives the qualitative reaction of sulfate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) is dissolved with 20 mL of water. The
resulting solution shall be red and clear.
ii. Lead: 2.0 g (1.95~2.04 g) of this product is weighed and dissolved with 50 mL ofwater and 5 mL of dilute hydrochloric acid, transferred to a 100 mL volumetric flask,
and added with water to the graduation line to make 100 mL. 50 mL of this solution ismeasured using a volumetric pipette to prepare a sample solution. When lead is tested
using the sample solution by the lead test method (dithizone method), the amount oflead shall be 10 mg/kg or less.
iii. Arsenic: 4.0 g (3.95~4.04 g) of this product is weighed, dissolved with 50 mL ofwater and 5 mL of dilute hydrochloric acid, transferred to a 100 mL volumetric flask,
and added with water to the graduation line to make 100 mL. 10 mL of this solution ismeasured using a volumetric pipette to make a sample solution. When arsenic is tested
using the sample solution by the method using device B, the color of mercuric bromide
paper shall not be darker than the standard color (5 mg/kg or less).
Assay: Appropriately 0.5 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is transferred to a 100 mL volumetric flask, dissolved with 50 mL of water,
and added with more water to the graduation line to make 100 mL. 10 mL of this solution
is measured using a volumetric pipette, added with 100 mL of water, and titrated with
0.01 mol/L ethylenediaminetetraacetic acid disodium solution, while adjusting to pH 8.0
with ammonia test solution (indicator: 0.2 g of murexide indicator).
0.01 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL

465

Provisional Translation from Japanese Original= 2.811 mg CoSO4ꞏ7H2O(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of cobalt sulfate(crystal) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of cobalt sulfate(crystal) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of and cobalt
sulfate (crystal) fillers.
Content: When this product is determined, it contains cobalt sulfate (CoSO4 7H2O)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 1 g of the
raw material for manufacturing of cobalt sulfate (crystal) is weighed, added with 100
mL of water, shaken up well, and filtered. To the filtrate the confirmation test i. for the
raw material for manufacturing of cobalt sulfate (crystal) is applied mutatis mutandis.
ii. According to the amount of this product on the label, the amount containing 1 g of the
raw material for manufacturing of cobalt sulfate (crystal) is weighed, added with 10
mL of water, shaken up well, and filtered. To the filtrate the confirmation test ii. forthe raw material for manufacturing of cobalt sulfate (crystal) is applied mutatis
mutandis.
Assay: The amount of this product containing approximately 0.5 g of cobalt sulfate (crystal)(CoSO4 7H2O) is weighed to three significant digits and the value is recorded. It is addedwith 20 mL of water, shaken up well, and filtered. The residue on the filter paper iswashed four times with 10 mL of water each time. The filtrate and the washings are
mixed, transferred to a 100 mL volumetric flask, and added with water to the graduationline to make 100 mL. The assay for the raw material for manufacturing of cobalt sulfate(crystal) is applied mutatis mutandis.
0.01 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL
= 2.811 mg CoSO4 7H2O

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Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of cobalt sulfate(crystal) is applied mutatis mutandis.

(101) Ferrous Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 80.0 % or more of ferrous sulfate(FeSO4).
Physical and chemical properties:
i. This product is grayish white powder with no odor.
ii. This product is easy to dissolve in newly boiled and cooled water, and hardly dissolves
in ethanol.
iii. This product is hygroscopic.
Confirmation test: A solution of this product in water (1 → 50) gives the qualitative
reactions of ferrous salt and sulfate.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is placed in a porcelain dish, dissolved
with 5 mL of aqua regia, and evaporated to dryness on a water bath. The residue isdissolved with 5 mL of 6 mol/L hydrochloric acid test solution and transferred to a
separatory funnel. The porcelain dish is washed twice with 5 mL each of 6 mol/Lhydrochloric acid test solution and the washings are put in the separatory funnel. The
mixed solution is shaken up twice with 40 mL of ether, then shaken up with 20 mL ofether, and allowed to stand and the separated ether layer is eliminated. The aqueous
layer is dissolved with 0.08 g (0.075~0.084 g) of hydroxylamine hydrochloride, heatedon a water bath for 10 minutes, allowed to cool, added with strong ammonia solution
by adding to adjust the pH of the solution to 3 to 4, and added with water to make 50
mL. This is used as a sample solution. 4.0 mL of the lead standard solution is put in aporcelain dish, added with 5 mL of aqua regia, and subjected to the same procedure as
for the sample solution. This is used as a control solution. When heavy metal is tested
using the control solution, the color of the sample solution shall not be darker than thatof the standard solution. However, if precipitation is generated when the pH of thesample solution is adjusted to 3~4, the pH is adjusted to 1.8~2.0, and the same test ishereinafter performed (40 mg/kg or less).

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Provisional Translation from Japanese Original

ii. Arsenic: 0.6 g (0.55~0.64 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (3.3 mg/kg or less)
Loss on drying: Less than 2.0 % (5 g, 105 °C, 2 hours)
Assay: Approximately 0.4 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 20 mL of water and 20 mL of dilute sulfuric acid, addedwith 2 mL of phosphoric acid, and titrated with 0.02 mol/L potassium permanganate
solution within 30 seconds.
0.02 mol/L potassium permanganate solution 1 mL = 15.19 mg FeSO4(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of ferrous sulfate(dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ferrous sulfate(dry) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of andferrous sulfate (dry) fillers.
Content: When this product is determined, it contains ferrous sulfate (FeSO4) corresponding
to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of ferrous sulfate (dry) is weighed,added with 20 mL of acetone and 30 mL of water, shaken up well, and filtered. To thefiltrate the confirmation test for the raw material for manufacturing of ferrous sulfate(dry) is applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.4 g of ferrous sulfate(FeSO4) is weighed to three significant digits and the value is recorded. It is added with
10 mL of acetone, 20 mL of water and 20 mL of dilute sulfuric acid, shaken up well, andfiltered. The residue is shaken up three times with 10 mL of water each time, and filteredusing the same filter paper. The filtrates are mixed, added with 2 mL of phosphoric acid,468

Provisional Translation from Japanese Originaland subjected to the assay for the raw material for manufacturing of ferrous sulfate (dry)is applied mutatis mutandis.
0.02 mol/L potassium permanganate solution 1 mL = 15.19 mg FeSO4(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of ferrous sulfate(dry) is applied mutatis mutandis.

(102) Copper Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 85.0 % or more of copper sulfate(CuSO4).
Physical and chemical properties:
i. This product is blue-white crystalline powder with no odor.
ii. This product is easy to dissolve in water, hard to slightly dissolve in glycerin, andhardly dissolves in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 3.0~4.5.
iv. This product is hygroscopic.
Confirmation test: A solution of this product in water (1 → 10) gives the qualitative
reactions of cuprate and sulfate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be blue and clear.
ii. Lead: 1.0 g (0.95~1.04 g) of this product is weighed and added with water to make 20
mL. This is used as a sample solution. 1.0 mL of the lead standard solution ismeasured, added with water to make 20 mL. This is used as a standard solution. Whenlead is tested using these sample and standard solutions by the lead test method(Method No. 1 of the atomic absorption spectrophotometry), the amount of lead shallnot be 10 mg/kg or less.
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed, dissolved with 20 mL ofwater, added with 4 mL of glacial acetic acid and 3 g (2.5~3.4 g) of potassium iodide,added with 20 mL of water, shaken up, allowed to stand for 5 minutes, filtered, andwashed with 5 mL of water. The filtrate and the washings are mixed and concentrated
by heating until the mixed solution reaches approximately 10 mL. After cooling, it isdissolved with 0.4 g (0.35~0.44 g) of ascorbic acid to prepare a sample solution. When469

Provisional Translation from Japanese Original

arsenic is tested using the sample solution by the method using device B, the color of
the sample solution shall not be darker than that of a solution prepared in the procedurein which 30 mL of water is added with 4 mL of glacial acetic acid, 3 g (2.5~3.4 g) ofpotassium iodide and 5.0 mL of arsenic standard solution, allowed to stand for 5
minutes, and hereinafter subjected to the same procedure as for the sample solution (10mg/kg or less).
Assay: Approximately 0.4 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 50 mL of water, and added with 4 mL of acetic acid and 3g (2.5~3.4 g) of potassium iodide. Then, the released iodine is titrated with 0.1 mol/L
sodium thiosulfate solution (indicator: 1 mL of starch test solution). A blank test is
performed in the same manner and corrections are made.
0.1 mol/L sodium thiosulfate solution 1 mL = 15.96 mg CuSO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of copper sulfate (dry)are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of copper sulfate(dry) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of andcopper sulfate (dry) fillers.
Content: When this product is determined, it contains copper sulfate (CuSO4) corresponding
to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of copper sulfate (dry) is weighed,added with 10 mL of water, shaken up well, and filtered. To the filtrate the confirmation
test for the raw material for manufacturing of copper sulfate (dry) is applied mutatis
mutandis.
Assay: The amount of this product containing approximately 0.4 g of copper sulfate(CuSO4) is weighed to three significant digits and the value is recorded. It is dissolved
with 50 mL of water, or dissolved with 20 mL of water as appropriate, then filtered andwashed with water. The filtrate and the washings are mixed to make 50 mL, and the assay

470

Provisional Translation from Japanese Originalfor the raw material for manufacturing copper sulfate (dry) is hereinafter applied mutatis
mutandis.
0.1 mol/L sodium thiosulfate solution 1 mL = 15.96 mg CuSO4(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of copper sulfate(dry) is applied mutatis mutandis.

(103) Copper Sulfate (crystal)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.5 % or more of copper sulfate(CuSO4 5H2O).
Physical and chemical properties:
i. This product is blue crystals, blocks or powder with no odor.
ii. This product is easy to dissolve in water and glycerin and hard to dissolve in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 2.5~4.0.
iv. This product is efflorescent.
Confirmation test: A solution of this product in water (1 → 10) gives the qualitative
reactions of cuprate and sulfate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be blue and clear.
ii. Lead: 1.0 g (0.95~1.04 g) of this product is weighed and added with water to make 20
mL. This is used as a sample solution. 1.0 mL of the lead standard solution ismeasured, added with water to make 20 mL. This is used as a standard solution. Whenlead is tested using these sample and standard solutions by the lead test method(Method No. 1 of the atomic absorption spectrophotometry), the amount of lead shallnot be 10 mg/kg or less.
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and dissolved with water tomake 25 mL. The purity test (3) for the raw material for manufacturing of copper
sulfate (dry) is applied mutatis mutandis (5 mg/kg or less).
Assay: Approximately 0.5 g of this product is weighed to the digits of 0.001 g and the value
is recorded, and the assay for the raw material for manufacturing of copper sulfate (dry) is
hereinafter applied mutatis mutandis.
0.1 mol/L sodium thiosulfate 1 mL = 24.97 mg CuSO4 5H2O

471

Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation (part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of copper sulfate(crystal) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of copper sulfate(crystal) is applied mutatis mutandis.
C. Preparation (part 2)(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of andcopper sulfate (crystal) fillers.
Content: When this product is determined, it contains copper sulfate (CuSO4 5H2O)
corresponding to 90~110 % of the amount on the label.
Confirmation test: According to the amount of this product on the label, the amountcontaining 1 g of the raw material for manufacturing of copper sulfate (crystal) is
weighed, added with 10 mL of water, shaken up well, and filtered. To the filtrate theconfirmation test for the raw material for manufacturing of copper sulfate (crystal) is
applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.5 g of copper sulfate(CuSO4 5H2O) is weighed to three significant digits and the value is recorded. It isdissolved with 50 mL of water, or dissolved with 20 mL of water as appropriate, filtered,and washed with water. The filtrate and the washings are mixed to make 50 mL and theassay for the raw material for manufacturing of copper sulfate (crystal) is hereinafter
applied mutatis mutandis.
0.1 mol/L sodium thiosulfate 1 mL = 24.97 mg CuSO4·5H2O(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of copper sulfate(crystal) is applied mutatis mutandis.

(104) Sodium Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards

472

Provisional Translation from Japanese OriginalContent: When this product is determined following a 4-hour drying at 105 °C, it contains
99.0 % or more of sodium sulfate (Na2SO4).
Physical and chemical properties:
i. This product is white powder with no odor.
ii. This product is easy to dissolve in water and hardly dissolves in ethanol.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of sodium salt and sulfate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 10
mL of newly boiled and cooled water. The resulting solution shall be colorless and its
turbidity shall be almost clear or clear.
ii. Heavy metals: 2.0 g (1.95~2.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (2 mg/kg or less).
Loss on drying: Less than 11.4 % (2 g, 105 °C, 4 hours)
Assay: This product is dried at 105 °C for 4 hours, approximately 0.4 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved with 200 mL of water,
added with 1 mL of hydrochloric acid, boiled, and gradually added with 8 mL of hot
barium chloride test solution. The resulting solution is heated on a water bath for an hour
and allowed to cool. The precipitation is collected by filtration, washed with water until
the washings produce no opacity when silver nitrate test solution is added to thewashings, dried, and ignited to the constant weight. Then the mass is weighed, which isregarded as the amount of barium sulfate (BaSO4).
Amount of sodium sulfate (Na2SO4) (mg)
= Amount of barium sulfate (BaSO4) (mg) × 0.6086(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards

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Provisional Translation from Japanese Original

The compositional standards of the raw material for manufacturing of sodium sulfate(dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium sulfate(dry) is applied mutatis mutandis.

(105) Magnesium Sulfate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 3-hour igniting at 450 °C, it contains
99.0 % or more of sodium sulfate (MgSO4).
Physical and chemical properties:
i. This product is white powder or crystalline powder with salty and bitter tastes.
ii. This product is easy to dissolve in water, hard to dissolve in glycerin and hardly
dissolves in ethanol.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of magnesium salt and sulfate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 10
mL of water. The resulting solution shall be colorless and its turbidity shall bedelicately slight cloudiness or less.
ii. Chloride: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.4 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.014 % or less).
iii. Heavy metals: 2.0 g (1.95~2.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Ignition residue: Less than 35.0 % (1 g, 450 °C, 3 hours)

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Provisional Translation from Japanese Original

Assay: This product is ignited at 450 °C for 3 hours, approximately 0.6 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved with 2 mL of dilutehydrochloric acid and water, transferred to a 100 volumetric flask, and added with morewater to the graduation line to make 100 mL. 25 mL of this solution is measured using a
volumetric pipette, added with 50 mL of water, 5 mL of pH 10.7 ammonia-ammonium
chloride buffer, and titrated with 0.05 mol/L ethylenediaminetetraacetic acid disodium
solution (indicator: 0.04 g (0.035~0.044 g) of eriochrome black T-sodium chloride
indicator). In this case, the end of titration is the time when the color of the solutionchanges from reddish violet to blue. A blank test is performed in the same way andcorrections are made.
0.05 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 6.019 mg
MgSO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of magnesium sulfate(dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of magnesium
sulfate (dry) is applied mutatis mutandis.

(106) Magnesium Sulfate (crystal)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 2-hour drying at 105 °C and 3-hour
igniting at 450 °C, it contains 99.0 % or more of sodium sulfate (MgSO4).
Physical and chemical properties:
i. This product is colorless pillar or needle crystals with salty and bitter tastes.
ii. This product is easy to dissolve in water or glycerin and hard to slightly dissolve inethanol.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of magnesium salt and sulfate.
Purity test:

475

Provisional Translation from Japanese Originali. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 10
mL of water. The resulting solution shall be colorless and its turbidity shall be almost
clear or clear.
ii. Chloride: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.4 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.014 % or less).
iii. Heavy metals: 2.0 g (1.95~2.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (10 mg/kg or less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of absorbing solution shall not be darker than
the standard color (4 mg/kg or less).
Ignition residue: 40.0~52.0 % (1 g, igniting at 450 °C for 3 hours after drying at 105 °C for
2 hours)
Assay: The compositional standards of the raw material for manufacturing of magnesium
sulfate (dry) are applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of magnesium sulfate(crystal) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of magnesium
sulfate (crystal) is applied mutatis mutandis.

(107) Manganese Sulfate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following 2-hour igniting at 450 °C, it contains
95.0 % or more of manganese sulfate (MnSO4).

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Physical and chemical properties:
i. This product is pink crystals or reddish white power with no odor.
ii. This product is easy to dissolve in water and hardly dissolves in ethanol.
iii. The pH of a solution of this product in water (1 → 20) is 4.5~6.5.
Confirmation test: A solution of this product in water (1 → 20) gives the qualitative
reactions of manganese salt and sulfate.
Purity test:
i. Clarity and color of solution: 2.0 g (1.95~2.04 g) of this product is dissolved with 20
mL of water and 0.5 mL of dilute hydrochloric acid. The resulting solution shall beclear.
ii. Lead: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved with nitric acid (1 →150), transferred to a 20 mL volumetric flask, and added with more nitric acid (1 →150) to the graduation line to make 20 mL. When lead is tested, using this solution as asample solution, by the lead test method (Method No. 1 of the atomic absorption
spectrophotometry), the amount of lead shall be 10 mg/kg or less.
iii. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (5 mg/kg or less)Ignition residue: Less than 42.0 % (1 g, 450 °C, 2 hours)
Assay: This product is ignited at 450 °C for 2 hours, approximately 0.1 g of it is weighed tothe digits of 0.001 g, and the value is recorded. It is dissolved with 20 mL of water,transferred to a 100 mL volumetric flask, and added with more water to the graduationline to make 100 mL. 30 mL of this solution is measured using a volumetric pipette, andadded with 70 mL of water, 5 mL of 0.1 mol/L magnesium-ethylenediaminetetraacetic
acid disodium solution and 0.1 g (0.05~0.14 g) of hydroxylamine hydrochloride. Then theassay for manganese carbonate is hereinafter applied mutatis mutandis.
0.01 mol/L ethylenediaminetetraacetic acid disodium solution 1 mL = 1.510 mg
MnSO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of manganese sulfate
are applied mutatis mutandis.
(b) Standard of storage method

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Provisional Translation from Japanese Original

The standard of storage method of the raw material for manufacturing of manganese
sulfate is applied mutatis mutandis.

(108) L-lysine Sulfate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 62.5 % or more of L-lysine sulfate((C6H14N2O2)2 H2SO4).
Physical and chemical properties:
i. This product is pale brown to pale brownish-red granules with a specific odor.
ii. A suspension of this product in water (1 → 10) is centrifuged. The pH of the obtained
supernatant is 4.5~7.5.
Confirmation test:
i. A suspension of this product in water (1 → 500) is centrifuged. 5 mL of the obtained
supernatant is added with 1 mL of ninhydrin test solution, heated for 3 minutes, addedwith 20 mL of water, and allowed to stand for 15 minutes. The resulting solution isreddish violet.
ii. A suspension of this product in water (1 → 50) is centrifuged. The obtained
supernatant gives the qualitative reaction i. of sulfate.
Purity test:
i. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is placed in a decomposition flask, addedwith 10 mL of nitric acid and 5 mL of sulfuric acid, and gently heated. If the solution
is still brown, it is allowed to cool, then is heated with 1~2 mL of additional nitricacid. This procedure is repeated until the solution becomes colorless to slightly yellow.
It is allowed to cool, added with 0.5 mL of perchloric acid, and heated until whitesmoke emerges. It is allowed to cool, added with 15 mL of saturated ammonium
oxalate solution and again heated until white smoke emerges. It is allowed to cool andadded with water to make approximately 10 mL. This is used as a sample solution.
When arsenic is tested by the method using device A, the color of the absorbing
solution shall not be darker than the standard color (2 mg/kg or less).

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Water content: Less than 5.0 % (direct titration)
Ignition residue: Less than 5.0 % (1 g)Assay: Approximately 45 mg of this product is weighed to the digits of 0.1 mg and thevalue is recorded. It is dissolved with 1 mol/L hydrochloric acid, transferred to a 50 mL
volumetric flask, and added with another 1 mol/L hydrochloric acid to the graduation line
to make 50 mL. 2.5 mL of this solution is measured using a volumetric pipette,
transferred to a 50 mL volumetric flask, added with 2.5 mL of L-norleucine test solution
using a volumetric pipette, dissolved with sodium citrate buffer (pH 2.2), added withmore sodium citrate buffer (pH 2.2) to the graduation line to make 50 mL, and filteredwith a membrane filter (0.45 µm). The filtrated is used as a sample solution. Separately,
24 mg of lysine hydrochloride is weighed to the digits of 0.01 mg and the value isrecorded. It is dissolved with 1 mol/L hydrochloric acid, transferred to a 50 mL
volumetric flask, and added with more 1 mol/L hydrochloric acid to the graduation line to
make 50 mL. 2.5 mL of this solution is measured using a volumetric pipette, transferred
to a 50 mL volumetric flask, added with 2.5 mL of L-norleucine test solution using a
volumetric pipette, dissolved with sodium citrate buffer (pH 2.2), and added with moresodium citrate buffer (pH 2.2) to the graduation line to make 50 mL. This is used as astandard solution. As for 100 µL each of the sample and standard solutions, an aminoacid automatic analyzer is operated under the following conditions, and a test is
performed by the internal standard method of the liquid chromatography.
Content of L-lysine sulfate (%) =

WS
AT
AiS
×
×
× 106.89
WT AiTAS

WT: Collected amount of the sample (mg)

WS: Collected amount of the reference standard (mg)
AT: Peak area of L-lysine in the sample solution
AiT: Peak area of L-norleucine in the sample solution
AS: Peak area of L-lysine in the standard solution
AiS: Peak area of L-norleucine in the standard solution
Operating conditions:
Detector: Visible absorption spectrometer (measurement wavelength: 570 nm)
Column: A stainless tube (inner diameter: approx. 4.0 mm, length: approx. 120mm) is filled with strong acid cation exchange resin.
Column temperature: 35 °C (0 → 15.3 min.) retention, 64 °C (15.3 → 31.0 min.)retention, 44 °C (31.0 → 44.4 min.) retention, 72 °C (44.4 →80.0 min.) retention
Reaction vessel temperature: Constant temperature at around 135 °C

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Mobile phase: Lithium citrate buffer
Reaction solution: Ninhydrin test solution for amino acid analysis
Flow rate of mobile phase: 0.50 mL/min.
Flow rate of reaction solution: 0.30 mL/min.
Column selection: A column to be used is selected when 100 µl of the standard
solution is operated under the above conditions, the internal
standard solution and L-lysine are eluted in that order, and theseparation degree is 5 or over.
(b) Standard of manufacturing method
For manufacturing, the lysine producing strain of Corynebacterium glutamicum isaerobically cultured. After the cultivation, the bacterial cells in the culture solution are heattreated, dried, and made into granules.
(c) Standard of storage method
It shall be put in a capped container and stored in a dry cold place.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of L-lysine sulfate areapplied mutatis mutandis.
(b) Standard of manufacturing method
The standard of manudacturing method of the raw material for manufacturing of L-lysine
sulfate is applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of L-lysine sulfate
is applied mutatis mutandis.

(109) Dipotassium hydrogen phosphate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following a 4-hour drying at 105 °C, it contains
98.0 % or more of dibasic potassium phosphate (K2HPO4).
Physical and chemical properties:
i. This product is white crystals or blocks.
ii. This product is easy to extremely dissolve in water and hard to slightly dissolve inethanol.
Confirmation test:480

Provisional Translation from Japanese Originali. When a solution of this product in water (1 → 20) is added with a drop ofphenolphthalein test solution, it is red.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions ofpotassium salt and phosphate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and its turbidity shall be delicate
slight cloudiness or less.
ii. Primary salt and tertiary salt: When 2.0 g (1.95~2.04 g) of this product is weighed,added with 50 mL of water, boiled for 5 minutes, cooled down to approximately
25 °C, and added with 3 drops of phenolphthalein test solution, it shall be red. When it
is added with 1.8 mL of 1 mol/L hydrochloric acid, it shall be colorless.
iii. Chloride: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.3 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.01 % or less).
iv. Sulfate: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the sulfate test method. A control solution is prepared using 0.4 mL of
0.005 mol/L sulfuric acid. When sulfate is tested, the opacity of the sample solution
shall not be higher than that of the control solution (0.02 % or less).
v. Carbonate: When 2.0 g (1.95~2.04 g) of this product is weighed, dissolved in 10 mL of
newly boiled and cooled water, and added with 5 mL of dilute hydrochloric acid, it
shall not bubble.
vi. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and neutralized withdiluted acetic acid and a sample solution is prepared by Method No. 1 of the heavy
metals test method. A control solution is prepared using 2.0 mL of lead standard
solution. When heavy metal is tested using these solutions, the color of the sample
solution shall not be darker than that of the control solution (20 mg/kg or less).
vii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: Less than 5.0 % (1 g, 105 °C, 4 hours)
Assay: This product is dried at 105 °C for 4 hours, approximately 3.0 g of it is weighed tothe digits of 0.01 g, and the value is recorded. It is dissolved with 50 mL of water andtitrated with 1 mol/L hydrochloric acid, maintaining it at approximately 15 °C (indicator:

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Provisional Translation from Japanese Original

3 to 4 drops of methyl orange-xylene cyanol FF test solution). In this case, the end of
titration is the time when the color of the solution changes from green to dark greenish
reddish violet. A blank test is performed in the same way and corrections are made.
1 mol/L hydrochloric acid 1 mL = 174.2 mg K2HPO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of dipotassiumhydrogen phosphate (dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of dipotassiumhydrogen phosphate (dry) is applied mutatis mutandis.

(110) Sodium Hydrogen Phosphate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 18.0~22.0 % of phosphorus (P) and27.0~32.5 % of sodium (Na).
Physical and chemical properties:
i. This product is white or gray crystalline powder with no odor.
ii. This product is easy to extremely dissolve in water and hardly dissolves in ethanol.
Confirmation test:
i. When a solution of this product in water (1 → 20) is added with a drop ofphenolphthalein test solution, it is red.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions i. and ii. of
sodium salt and the qualitative reaction of phosphate.
Purity test:
i. Arsenic: 0.166 g (0.1655~0.1664 g) of this product is weighed and dissolved with 5mLof dilute hydrochloric acid and the sample solution is prepared by method No. 1 of thearsenic test method. The arsenic test is performed by the method using device A. The
color of the absorbing solution shall not be darker than the standard color (12 mg/kg or
less).
ii. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed, added with dilutehydrochloric acid heated to dissolve, allowed to cool, and added with water to make 50482

Provisional Translation from Japanese Original

mL. This is used as a sample solution. A control solution is prepared using 5.0 mL oflead standard solution. When heavy metal is tested using these solutions, the color of
the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
iii. Fluorine: 1.0 g (0.95~1.04 g) of this product is weighed and added withapproximately 0.2~0.3 g of silicic acid powder, and steam distillation is performed.
The distillate liquid is neutralized with sodium hydroxide solution (1 → 100)
(indicator: methyl red test solution), transferred to a 250 mL volumetric flask, and is
added with water to the graduation line to make 250 mL. 5 mL of this solution ismeasured using a volumetric pipette, transferred to a 25 mL volumetric flask, addedwith 5 mL of water, 3 mL of lanthanum-alizarin complexone test solution and 10 mLof acetone, allowed to cool, and is added with water to the graduation line to make 25
mL. This is used as a sample solution. Separately, 5 mL of fluorine standard solution ismeasured using a volumetric pipette, added with 5 ml of water, and hereinafter
subjected to the same procedure as for the sample solution. This is used as a standard
solution. Separately, 5 mL of water is measured and the same procedure is performed
to prepare a blank test solution. The sample and standard solutions are separately
shaken up and allowed to stand for 90 minutes. When using the blank test solution as acontrol solution, the absorbance is measured at the absorption maximum at awavelength around 620 nm, the absorbance of the sample solution shall be that or lessof the standard solution (0.125 % or less).
Loss on drying: Less than 2.0 % (1 g, 105 °C, 2 hours)
Assay:
i. Phosphorus: Approximately 2.0 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is added with 50 mL of dilute hydrochloric acid, dissolved bywarming, allowed to cool, transferred to a 250 mL volumetric flask, added with water
to the graduation line to make 250 mL, and filtered with dry filter paper. 20 mL of thefirst filtrate is eliminated and 10 mL of the next filtrate is measured using a volumetricpipette, transferred to a 100 mL volumetric flask, and added with water to thegraduation line to make 100 mL. 10 mL of this solution is measured using a
volumetric flask, and transferred to a 100 mL volumetric flask. When it is strongly
acidic, it is neutralized with ammonia test solution using phenolphthalein as anindicator and make it slightly acid by the addition of several drops of nitric acid, addedwith water to make 70 mL, and added with 20 mL of phosphorus coloring test solution
and water to the graduation line to make 100 mL. This is used as a sample solution.
Separately, 10 mL and 20 mL of phosphorus standard solution are measured using a

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Provisional Translation from Japanese Original

volumetric pipette. Each of them is transferred to each 100 mL volumetric flask, addedwith water to make approximately 70 mL, added with 20 mL of phosphorus coloring
test solution and water to the graduation line to make 100 mL, and used as the S1 andS2 solutions, respectively. The absorbances AT and AS for the sample solution and S2
solution, respectively, are measured at the absorption maximum at a wavelengtharound 400~420 nm, using S1 solution as a control solution.
Amount of phosphorus (P) (mg) = 1 (mg) +

AT
(mg)
AS

ii. Sodium: Approximately 2.0 g of this product is weighed to the digits of 0.01 g and thevalue is recorded. It is added with 50 mL of dilute hydrochloric acid, dissolved bywarming, allowed to cool, transferred to a 250 mL volumetric flask, added with water
to the graduation line to make 250 mL, and filtered with dry filter paper. 20 mL of thefirst filtrate is eliminated. 5 mL of the next filtrate is measured using a volumetricpipette, transferred to a 500 mL volumetric flask, and is added with water to thegraduation line to make 500 mL. This is used as a sample solution. Separately, 20, 25
and 30 mL of sodium standard solutions are measured and each of them is transferred
to a 1 L volumetric flask and is added with water to the graduation line to make 1 L.
They are used a standard solution. For the sample and standard solutions, the intensityof light at a wavelength of 589 nm is measured using a flame spectrophotometer, andthe amount of sodium (Na) in the sample solution is calculated from the calibration
curve obtained from the standard solutions. Propane and air are used as fuel gas andsupporting gas, respectively, of a flame spectrophotometer.
(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium hydrogen
phosphate (dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Sodiumhydrogen phosphate (dry) is applied mutatis mutandis.

(111) Potassium Dihydrogen Phosphate (dry)A. Raw material for manufacturing(a) Compositional standards

484

Provisional Translation from Japanese OriginalContent: When this product is determined following a 4-hour drying at 105 °C, it contains
98.0 % or more of potassium dihydrogen phosphate (KH2PO4).
Physical and chemical properties:
i. This product is colorless crystals or white crystalline powder.
ii. This product is easy to dissolve in water and hardly dissolves in ethanol.
Confirmation test:
i. A solution of this product in water (1 → 20) is acidic.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions ofpotassium salt and phosphate.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and its turbidity shall be delicate
slight cloudiness or less.
ii. pH: The pH of a solution of this product in water (2.7 → 100) shall be 4.2~4.7.
iii. Chloride: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.3 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.01 % or less).
iv. Sulfate: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the sulfate test method. A control solution is prepared using 0.4 mL of
0.005 mol/L sulfuric acid. When sulfate is tested, the opacity of the sample solution
shall not be higher than that of the control solution (0.02 % or less).
v. Heavy metals: 0.5 g (0.45~0.54 g) of this product is weighed and neutralized withdiluted acetic acid and a sample solution is prepared by Method No. 1 of the heavy
metals test method. A control solution is prepared using 1.0 mL of lead standard
solution. When heavy metal is tested using these solutions, the color of the sample
solution shall not be darker than that of the control solution (20 ppm or less).
vi. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 ppm or less).Loss on drying: Less than 0.5 % (1 g, 105 °C, 4hours)Assay: This product is dried at 105 °C for 4 hours, approximately 3.0 g of it is weighed tothe digits of 0.01 g, and the value is recorded. It is dissolved with 30 mL of water, addedwith 5 g (4.5~5.4 g) of sodium chloride, dissolved by shaking well, and titrated with 1
mol/L sodium hydroxide solution, maintaining it at approximately 15 °C (indicator: 3~4

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Provisional Translation from Japanese Original

drops of thymol blue test solution). In this case, the end of titration is the time when thecolor of the solution changes from yellow to blue. A blank test is performed in the samemanner and corrections are made.
1 mol/L sodium hydroxide solution 1 mL = 136.1 mg KH2PO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of potassium
dihydrogen phosphate (dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
dihydrogen phosphate (dry) is applied mutatis mutandis.

(112) Sodium Dihydrogen Phosphate (dry)A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried and determined, it contains 98.0~102.0 % of sodium
dihydrogen phosphate (NaH2PO4).
Physical and chemical properties:
i. This product is white powder or particles.
ii. This product is easy to extremely dissolve in water and hardly dissolves in ethanol.
Confirmation test:
i. A solution of this product in water (1 → 20) is weak acid.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions i. and ii. of
sodium salt and the qualitative reaction of phosphate.
Purity test:
i. Clarity and color of solution: 2.0 g (1.95~2.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and its turbidity shall be delicate
slight cloudiness or less.
ii. Chloride: 0.2 g (0.15~0.24 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.6 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.1 % or less).

486

Provisional Translation from Japanese Originaliii. Sulfate: 0.5 g (0.45~0.54 g) of this products is weighted and a sample solution isprepared by the sulfate test method. A control solution is prepared using 0.5 mL of
0.005 mol/L sulfuric acid. When sulfate is tested, the opacity of the sample solution
shall not be higher than that of the control solution (0.048 % or less).
iv. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: Less than 2.0 % (1 g, 105 °C, 5 hours)
Assay: This product is dried, approximately 3.0 g of it is weighed to the digits of 0.01 g, andthe value is recorded. It is dissolved in 30 mL of water, added with 5 g (4.5~5.4 g) of
sodium chloride, shaken up well to dissolve, and titrated with 1 mol/L sodium hydroxide
solution maintaining it at approximately 15 °C (indicator: 3~4 drops of thymol blue test
solution). In this case, the end of titration is the time when the color of the solutionchanges from yellow to blue. A blank test is performed in the same manner andcorrections are made.
1 mol/L sodium hydroxide solution 1 mL = 120.0 mg NaH2PO4(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium dihydrogen
phosphate (dry) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
dihydrogen phosphate (dry) is applied mutatis mutandis.

(113) Sodium Dihydrogen Phosphate (crystal)A. Raw material for manufacturing(a) Compositional standards

487

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains 98.0 % or more of sodium dihydrogen
phosphate (NaH2PO4 2H2O).
Physical and chemical properties:
i. This product is colorless to white crystals or white crystalline powder.
ii. This product is easy to dissolve in water and hardly dissolves in ethanol.
Confirmation test:
i. A solution of this product in water (1 → 20) is weak acid.
ii. A solution of this product in water (1 → 20) gives the qualitative reactions i. and ii. of
sodium salt and the qualitative reaction of phosphate.
Purity test:
i. Clarity and color of solution: 2.0 g (1.95~2.04 g) of this product is dissolved with 20
mL of water. The resulting solution shall be colorless and its turbidity shall be delicate
slight cloudiness or less.
ii. Chloride: 1.0 g (0.95~1.04 g) of this products is weighted and a sample solution isprepared by the chloride test method. A control solution is prepared using 0.3 mL of
0.01 mol/L hydrochloric acid. When chloride is tested, the opacity of the sample
solution shall not be higher than that of the control solution (0.01 % or less).
iii. Sulfate: 0.5 g (0.45~0.54 g) of this products is weighted and a sample solution isprepared by the sulfate test method. A control solution is prepared using 0.4 mL of
0.005 mol/L sulfuric acid. When sulfate is tested, the opacity of the sample solution
shall not be higher than that of the control solution (0.038 % or less).
iv. Heavy metals: 1.0 g (0.95~1.04 g) of this product is weighed and a sample solution isprepared by Method No. 1 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution. When heavy metal is tested using
these solutions, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
v. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 1 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: 22.0~24.0 % (1 g, 40 °C, 16 hours and 105 °C, 5 hours)
Assay: This product is dried, approximately 3.0 g of it is weighed to the digits of 0.01 g, andthe value is recorded. It is dissolved in 70 mL of water, added with 5 g (4.5~5.4 g) of
sodium chloride, shaken up well to dissolve, and the assay for the raw material for
manufacturing of sodium dihydrogen phosphate (dry) is hereinafter applied mutatis
mutandis.

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Provisional Translation from Japanese Original

1 mol/L sodium hydroxide solution 1 mL = 156.0 mg NaH2PO4 2H2O(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium dihydrogen
phosphate (crystal) are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
dihydrogen phosphate (crystal) is applied mutatis mutandis.
(114) Zinc Bacitracin
A. Raw material for manufacturing(a) Compositional standards
Potency: The potency test shows that 4.2 units of this product are contained in 1 mg.
Physical and chemical properties: This product is yellowish-gray-brown to brown powder
with a specific odor.
Confirmation test:
i. The amount of this product containing approximately 4,200 units of bacitracin isweighed, added with 100 mL of buffer No. 3, stirred for approximately 15 minutes andthen allowed to stand. The supernatant of the mixture is a sample solution. Separately,
the amount of working standard bacitracin containing approximately 4,200 units ofbacitracin is weighed, added with 1 g (0.5~1.4 g) of peptone, and dissolved in 100 mLof No. 3 buffer. This solution is used as a standard solution. 5 µl each of the sample
and standard solutions are spotted on a thin layer plate prepared using silica gel forthin-layer chromatography. Then, they are developed with the developing solvent,acetone (3 → 10), by the ascending method and the thin layer plate is air dried. The
thin layer plate is placed on a large flat plate medium consisting of a base layer(approximately 3 mm thick) and a inoculated layer (approximately 1 mm thick),allowed to stand for approximately 1 hour at room temperature, taken off, cultured at
37 °C for 16 hours, and then observed. The Rf values of the inhibition zones of thesample and standard solutions are equal.
Agar plate: Medium No. 1 is used for both the media of base and inoculated layers,
and 1 % of bacterial culture is added in the inoculated layer medium.

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Provisional Translation from Japanese Original

Preparation of bacterial culture: The bacterial culture is prepared using Micrococcus
luteus ATCC 10240 as a test strain by the potency test method specified in General
Tests.
ⅱ. 5 g (4.5~5.4 g) of this product is weighed, incinerated at 550 °C for 4 hours, addedwith 10 mL of 3 mol/L hydrochloric acid test solution, covered with a watch glass, andthen mildly heated for 10 minutes. It is allowed to cool, filtered, added with water tomake 50 mL, and neutralized with sodium hydroxide solution (1 → 5) (it is filtered if
necessary). This solution gives the qualitative reaction iii. of zinc salt.
Purity test:
i. pH: The pH of water suspension (1 → 10) of this product shall be 5.5~7.5.
ⅱ. Lead: Approximately 10.0 of this product is weighed to the digit of 0.1 g and the value
is recorded. It is incinerated at 550 °C for 4 hours, allowed to cool, added with 10 mLof hydrochloric acid (1 → 10), covered with a watch glass, and mildly heated for 10
minutes. It is allowed to cool and filtered. The filtrate is transferred to a 10 mL
volumetric flask and added with water to the marked line to make 10 mL. This is used
as a sample solution. Separately, 5 mL of lead standard solution for dithizone ismeasured using a volumetric pipette, transferred to a 10 mL volumetric flask, andadded with water to the marked line to make 10 mL. This is used as a standard
solution. The absorbance AT and AS of the sample and standard solutions are measured
at the wavelength of 283.3 nm using an atomic absorption spectrophotometer with alead hollow cathode lamp and the amount of lead in the sample is calculated by thefollowing formula. The amount shall be 10 mg/kg or less.
The amount of lead (mg/kg) = 0.5 ×

ATAS

iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: 12.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 35.0 % or less (1 g)
Nitrogen: 5.0~10.0 % (Kjeldahl method)
Crude fat: 10.0 % or lessCrude fiber: 7.0 % or lessPotency test:
Agar plate: No. 1 medium is used for both the media of base and inoculated layers.
Test strain: Micrococcus luteus ATCC 10240 is used.

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Provisional Translation from Japanese Original

Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is added with buffer No. 3 to make a constant volume at a concentration of
approximately 100 units/mL. This is used as a diluted stock solution. The volume ofthe diluted stock solution required for testing is measured using a volumetric pipette
and accurately diluted by adding buffer No. 3 to prepare the working standard diluent
at a high concentration of 2 units/mL and the working standard diluent at a low
concentration of 0.5 units/mL.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a mixed
solution of buffer No. 3 and pyridine (31:9), shaken or pretreated with approximately5
mL of 1 mol/L hydrochloric acid test solution to adjust pH to 2.0 or less, and stirred
for approximately 3 minutes. Then, it is added with a mixed solution of buffer No. 3
and pyridine (31:9) to make a constant volume with the concentration 100 units/mL(estimated value) and stirred for 15~30 minutes without foaming. The solution iscentrifuged or allowed to stand and the supernatant is used as a sample stock solution.
The volume of the sample stock solution required for testing is measured using a
volumetric pipette and accurately diluted by adding buffer No. 3 to prepare the sample
solution at a high concentration of 2 units/mL (estimated value) and the sample
solution at a low concentration of 0.5 units/mL (estimated value).
(b) Standard of manufacturing method
For manufacturing, the bacitracin producing strain of Bacillus licheniformis is aerobicallycultured. Bacitracin in the culture solution after the cultivation is used as a zinc complex
salt, and it is added with calcium carbonate as appropriate, and the culture solution is dried.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of zinc bacitracin are
applied mutatis mutandis.
(b) Standard of manufacturing method
The standard of manufacturing method of the raw material for manufacturing of zincbacitracin is applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc bacitracin
is applied mutatis mutandis.

491

Provisional Translation from Japanese Original(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture
C. Preparation (Part 2)(a) Compositional standards
This product is pieces or powder of the mixture of the raw material for manufacturing ofzinc bacitracin and carriers.
Potency: The potency test shows that it contains 85~125 % of the label potency.
Physical and chemical properties:
i. This product is pale yellow grayish white to brown small pieces or powder with a
specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:
i. The Confirmation test i. of the raw material for manufacturing of zinc bacitracin are
applied mutatis mutandis.
ⅱ. According to the label potency of this product, the amount containing approximately
21,000 units of bacitracin is weighed and incinerated at 550 °C for 4 hours. The
confirmation test ⅱ. of the raw material for manufacturing of zinc bacitracin ishereinafter applied mutatis mutandis.
Loss on drying: 15.0 % or less (1 g, 105 ℃, 3 hours)
Potency test:
Agar plate: The provisions of the raw materials for manufacturing of zinc bacitracin are
applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of zinc bacitracin are
applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of zinc bacitracin are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount of this product required for testing is weighed to three significant digits andthe value is recorded. It is added with a mixed solution of buffer No. 3 and pyridine(31:9), shaken or pretreated with approximately 5 mL of 1 mol/L hydrochloric acidtest solution to adjust pH to 2.0 or less, and stirred for approximately 3 minutes. Then,
it is added with a mixed solution of buffer No. 3 and pyridine (31:9) to make a

492

Provisional Translation from Japanese Original

constant volume at a concentration 100 units/mL or less, and the provisions of the raw
materials for manufacturing of zinc bacitracin are hereinafter applied mutatis
mutandis.
(b) Standard of manufacturing method
It shall be manufactured by mixing the raw materials for manufacturing of zinc bacitracinwith carriers.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of zinc bacitracin
is applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期限 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture(115) Avilamycin
A. Raw material for manufacturing(a) Compositional standards
Potency: The potency test shows that 100 µg (potency) of this produce are contained in 1
mg.
Physical and chemical properties: This product is gray to dark brown powder or particles
with a specific odor.
Confirmation test: The amount of this product containing approximately 100 mg (potency)of avilamycin is weighed, added with 10 mL of acetone, shaken for 10 minutes, andfiltered. This filtrate is used as a sample solution. Separately, the amount of the working
standard avilamycin containing approximately 10 mg (potency) of avilamycin is weighed
and dissolved with 1 mL of acetone. This is used as a standard solution. 10 µL each of thesample and standard solutions are spotted on a thin layer plate prepared using silica gelfor thin-layer chromatography. Then, they are developed to approximately 10 cm with thedeveloping solvent, a mixed solution of ethyl acetate, cyclohexane, and methanol(85:15:4), and the thin layer plate is air dried. It is sprayed with a mixed solution of
sulfuric acid and methanol (1:1) and heated at 100 °C for 5 minutes. Then, the Rf valuesof the main spots obtained from the sample and standard solutions are equal.

493

Provisional Translation from Japanese Original

Purity test:
i. pH: This product 1.0 g (0.95~1.04 g) is added with 20 mL of water, shaken for 10
minutes, and filtered. This pH of this filtrate shall be 5.0~8.5.
ⅱ. Heavy metal: 1.0 g of this product (0.95~1.04 g) is weighed. In the test using thesample solution prepared by method NO.3 of the heavy metal test method and thecontrol solution using 3.0 mL of lead standard solution, the color of the sample
solution shall not be darker than that of the control solution (30 mg/kg or less).
ⅲ. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
ⅳ. Content ratio of avilamycin A: This product 1.0 g (0.95~1.04 g) is weighed, addedwith 100 mL of acetone, and stirred for 1 hour. The supernatant 4 mL is measured, thesolvent is distilled away under reduced pressure in a water bath at 50 °C, and theresidue is dissolved with 5 mL of acetonitrile and then added with 5 mL of pH 7.0
phosphate buffer. The solution is filtered with a membrane filter (0.45 µm) and thefiltrate is used as a sample solution. When the sample solution 50 µL is tested by theliquid chromatography under the following conditions, the content ratio of avilamycin
A shall be 60 % or greater.
The content ratio of avilamaycin A in this product (%) =

AT1
× 100
AT

AT1: Peak area of avilamaycin A in the sample solution
AT: Sum of peak areas of the sample solution
Operating condition:
Detector: Ultraviolet absorptiometer (measurement wavelength: 286 nm)
Column: A stainless tube (internal diameter: 4.0~4.6 mm, length: 200~250 mm) isfilled with 5 µm of octadecyl-silylated silica gel.
Column temperature: Constant temperature around 25 °C
Mobile phase A: Mixed solution of methanol and pH 7.0 phosphate buffer (11:9)
Mobile phase B: Mixed solution of methanol and pH 7.0 phosphate buffer (4:1)
Gradient method: The mixing ratio of mobile phases A and B is 78:22. After 5
minute elution, the mixing ratio is changed to 35:65 in the linear gradient for 30
minutes, and then the mixing ratio is maintained.
Flow rate: The retention time of avilamaycin A is adjusted to approximately 25
minutes.

494

Provisional Translation from Japanese Original

Column selection: 5 mg each of Sec-butyl-p-hydroxybenzoate and butyl-phydroxybenzoate (4.5~5.4 mg) are dissolved in 50 mL of acetonitrile and addedwith water to make 100 mL. The column to be used: when 10 µL of this solution
is operated under the above conditions with the mixing ratio of mobile phases A
and B, 78:22, and sec-butyl-p-hydroxybenzoate and butyl-p-hydroxybenzoate are
eluted in that order, the separation degree shall be 3.0 or greater.
Area measurement range: About twice as much as the retention time of avilamaycin
A.
Loss on drying: 10.0 % or less (1 g, decompression, 100 ℃, 3 hours)
Ignition residue: 70.0 % or less (1 g)
Nitrogen: 6.0 % or less (kjeldahl method)
Crude fat: 20.0 % or lessCrude fiber: 20.0 % or lessPotency test:
Agar plate (single layer): 10 mL of buffer No. 4 mixed with the test strain (11 mL in a
Petri dish with internal diameter of 100 mm) is used.
The strain: Micrococcus luteus ATCC 10240 is used.
Preparation of working standard diluent: The amount of the working reference standard
required for testing is weighed to three significant digits and the value is recorded. It isdissolved with acetone, and is further added with acetone to make an accurate constantvolume at a concentration of approximately 1 mg/mL (potency). This is used as adiluted stock solution. The volume of the diluted stock solution required for testing ismeasured using a volumetric pipette and accurately diluted by adding a mixed solutionof acetone and buffer No. 7 (1:4) to prepare the working standard diluent at a highconcentration of 2 µg/mL (potency) and the working standard diluent at a low
concentration of 0.5 µg/mL (potency).
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added withapproximately 10

hold buffer No. 7 using a volumetric pipette and mildly shaken.

Next, it is added with acetone four times as much as buffer No. 7 using a volumetricpipette, stirred for 20 minutes, filtered or centrifuged. This is used as a sample stock
solution. The volume of the sample stock solution required for testing is measured
using a volumetric pipette and accurately diluted by adding a mixed solution ofacetone and buffer No. 7 (1:4) to prepare the sample solution at a high concentration of
2 µg/mL (estimated value) (potency) and the sample solution at a low concentration of
0.5 µg/mL (estimated value) (potency).

495

Provisional Translation from Japanese Original(b) Standard of manufacturing method
The avilamaycin producing strain of Streptomyces viridochromogenes is aerobicallycultured, and after the cultivation, where appropriate added with calcium hydroxide andothers as a coagulating agent to separate solids. The solids are dried.
(c) Standard of storage method
It shall be stored in a sealed container
B. Preparation(a) Compositional standards
This product is powder or particles of the mixture of the raw material for manufacturingof avilamycin and carriers.
Potency: The potency test shows that it contains 85~125 % of the label potency.
Physical and chemical properties:
i. This product is pale orange-yellow to brown or blackish brown power or particles witha specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test: The confirmation test of the raw material for manufacturing ofavilamycin are applied mutatis mutandis.
Loss on drying: 10.0 % or less (1 g, decompression, 100 ℃, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing of theavilamycin are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of the avilamycin are
applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of the avilamycin are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount of this product required for testing is weighed to three significant digits andthe value is recorded. It is added with approximately 10 hold buffer No. 7 using a
volumetric pipette and mildly shaken. Then, it is added with acetone four times asmuch as buffer No. 7 using a volumetric pipette, stirred for 20 minutes, and theprovisions of the raw materials for manufacturing of avilamaycin are hereinafter
applied mutatis mutandis.
(b) Standard of manufacturing method
It shall be manufactured by mixing the raw materials for manufacturing of avilamycin
with carriers.

496

Provisional Translation from Japanese Original(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of avilamycin isapplied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture(116) Enramycin
A. Raw material for manufacturing(a) Compositional standards
Potency: The potency test shows that 10.0 µg (potency) or more of this product arecontained in 1 mg.
Physical and chemical properties: This product is grayish brown to brown powder orparticles with a slightly specific odor.
Confirmation test:
i. The amount of this product containing approximately 10 mg (potency) of enramycin isweighed, and dissolved with 50 mL of a mixed solution of methanol and 0.1 mol/Lhydrochloric acid test solution (1:1), shaken for approximately 20 minutes, and thenfiltered. The filtrate is added with ammonia test solution to make it neutral to slightly
alkaline and filtered as appropriate to prepare a sample solution. The sample solution
20 mL is added with approximately 1 mL of porous styrene divinylbenzene copolymer
refined resin immersed in methanol (1 → 2), shaken for 10 minutes, and filtered with aglass filter. The resin is washed twice with approximately 20 mL of water, added with
20 mL of a mixed solution of methanol and 0.2 mol/L hydrochloric acid test solution(7:3), vigorously shaken, and filtered. In the measurement of the absorption spectrumof this solution, the absorption maximum and the absorption minimum are at the
wavelengths of 269~275 nm and 250~256 nm, respectively.
ⅱ. The amount of this product containing approximately 100 mg (potency) of enramycin
is weighed, added with 50 mL of a mixed solution of acetone, 1 mol/L hydrochloric
acid test solution and water (35:12:56), stirred, neutralized with ammonia test solution,and filtered or centrifuged. The volume required for testing of the filtrate or
supernatant is measured, added with acetone (7 → 20) to make its concentration
approximately 1 mg/mL (potency) to prepare a sample solution. 5 µL each of this497

Provisional Translation from Japanese Original

sample solution and the working standard diluted stock solution obtained by thepotency test are spotted on a thin layer plate prepared using cellulose powder for thinlayer chromatography. They are developed to approximately 10 cm using the upper
layer solution of a mixed solution of n-butanol, pyridine and water (8:3:9) as thedeveloping solvent and the thin layer plate is air-dried. When the thin layer plate isuniformly sprayed with the acetone solution of ninhydrin (1 → 50) and heated at
105 °C for 10 minutes, the main spot of the sample solution and the spot of theworking standard diluted stock solution are purple and their Rf values are equal.
Purity test:
i. pH: The pH of solution (1 → 10) of this product shall be 5.0~8.0.
ⅱ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by Method No. 3 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 3.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (30 mg/kg or less).
ⅲ. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
Loss on drying: 6.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Acid soluble ash: Approximately 1 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is added with 20 mL of dilute hydrochloric acid, heated at 50 °C
for 15 minutes while stirring, allowed to cool, placed in a 50 mL volumetric flask, addedwith water to the marked line to make 50 mL, and filtered. This solution is centrifugeduntil clear as appropriate and 25 mL of it is measured, added with 1 mL of dilute sulfuric
acid, evaporated to dryness, ignited to constant weight at 800 ± 25 °C. The resulting
weight shall be 20.0 % or less.
Nitrogen: 1.0~8.0 % (Kjeldahl method)
Crude fat: 20.0 % or lessCrude fiber: 5.0 % or lessPotency test:
Agar plate (single layer): 10 mL of the No. 13 medium mixed with the test strain is used
(11 mL in a Petri dish with internal diameter of 100 mm).
Test strain: Bacillus subtilis ATCC 6633 is used.
Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value is498

Provisional Translation from Japanese Originalrecorded. It is dissolved with methanol (4 → 5), and is further added with methanol (4→ 5) to make a constant volume at a concentration of approximately 1 mg/mL(potency). This is used as a diluted stock solution. The volume required for testing of
the diluted stock solution is measured using a volumetric pipette and accurately diluted
by adding buffer No. 8 to prepare the working standard diluent at a high concentrationof 40 µg/mL (potency) and the working standard diluent at a low concentration of 10µg/mL (potency).
Preparation of the sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a constantvolume of a mixed solution of acetone, 1 mol/L hydrochloric acid test solution andwater (35:12:56) using a volumetric pipette to make its concentration (estimated value)
approximately 1 mg/mL (potency) and stirred. It is confirmed to be pH 2.8~3.2, asappropriate the pH is adjusted with 1 mol/L hydrochloric acid test solution or 1 mol/L
sodium hydroxide test solution, and then filtered or centrifuged to prepare a sample
stock solution. The volume required for testing of the sample stock solution ismeasured using a volumetric pipette and accurately diluted by adding buffer No. 8 toprepare the sample solution at a high concentration (estimated value) of 40 µg/mL(potency) and the sample solution at a low concentration (estimated value) of 10µg/mL (potency).
(b) Standard of manufacturing method
The enramycin producing strain of Streptomyces fungicidicus is aerobically cultured, andafter cultivation the pH of the culture solution is adjusted. Then, the solution is filtered,
where appropriate using a filter aid, and the obtained solids are dried.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
B. Preparation(a) Compositional standards
This product is pieces or powder of the mixture of the raw material for manufacturing ofenramycin and carriers.
Potency: The potency test shows that it contains 85~125 % of the label potency.
Physical and chemical properties:
i. This product is grayish brown to brown or yellow brown small pieces or powder with aslightly specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.

499

Provisional Translation from Japanese OriginalConfirmation test: The confirmation test of the raw materials for manufacturing ofenramycin are applied mutatis mutandis.
Loss on drying: 10 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing ofenramycin are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of enramycin are
applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of enramycin are applied mutatis mutandis.
Preparation of the sample solution: Where appropriate, this product is ground, according
to the label potency, the amount required for testing is weighed to three significant
digits and the value is recorded. It is added with a constant volume of a mixed solutionof acetone, 1 mol/L hydrochloric acid test solution and water (35: 12: 56) using a
volumetric pipette to make its concentration approximately 1 mg/mL (potency),
stirred, and the provisions of the raw materials for manufacturing of enramycin are
hereinafter applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of enramycin is mixed withcarriers, and as appropriate particle-seized or sieved.
(c) Standard of storage method
The standard storage method of the raw materials for manufacturing of enramycin are
applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture(117) Salinomycin Sodium
Salinomycin sodium (part 1)
A. Raw material for manufacturing(a) Compositional standards
Potency: This product is sodium salt of salinomycin. The potency test shows that 800 µg(potency) or more is contained in 1 mg.

500

Provisional Translation from Japanese Original

Physical and chemical properties:
i. This product is white to pale yellow-white crystalline powder with a slightly specificodor.
ⅱ. This product is extremely easy to dissolve in ethyl acetate, easy to dissolves in
acetone, ethylether, chloroform and methanol, hard to lightly dissolve in n-hexane, andhardly dissolve in water.
Confirmation test:
i. This product in methanol solution (3 → 1,000) 2 mL is added with 2 mL of vanillinhydrochloric acid test solution and gently shaken. The resulting solution is red.
ⅱ. This product in methanol solution (1 → 10,000) 9 mL is added with 1 mL of 1 mol/Lhydrochloric acid test solution, shaken well, allowed to stand at room temperature for5 minutes. In the measurement of the ultraviolet absorption spectrum of this solution,
the absorption maximum is at the wavelengths of 285~300 nm.
iii. The solution obtained when 3 g (2.5~3.4 g) of this product is ashed (incinerated),shaken with 10 mL of water and filtered gives the qualitative reaction of sodium salt.
Purity test: The potency test shows that it contains 85~125 % of the label potency.
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is weighed, dissolved
with 20 mL of methanol. The resulting solution shall be pale yellow and clear or
almost clear.
ⅱ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 3 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
Loss on drying: 7.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 7.0~12.0 % (1 g)
Potency test:
Agar plate: Medium No. 14 is used for both the media of base and inoculated layers.
However, the medium of base layer is 10 mL (11 mL in a Petri dish with internal
diameter of 100 mm).
Test strain: Bacillus subtilis ATCC 6633 is used.

501

Provisional Translation from Japanese Original

Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is dissolved with methanol, and is further added with methanol to make aconstant volume at a concentration of approximately 400 µg/mL (potency). This isused as a diluted stock solution. The volume required for testing of the diluted stock
solution is measured using a volumetric pipette and accurately diluted by adding waterand methanol to prepare the working standard diluent at a high concentration of 40µg/mL (potency) with 10 % methanol and the working standard diluent at a low
concentration of 10 µg/mL (potency) with 10 % methanol.
Preparation of the sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved withmethanol to make an accurate constant volume with the concentration (estimatedvalue) of approximately 400 µg/mL (potency). This is used as a sample stock solution.
The volume required for testing of the sample stock solution is measured using a
volumetric pipette and accurately diluted by adding water and methanol to prepare thesample solution at a high concentration (estimated value) of 40 µg/mL (potency) and
10 % methanol and the sample solution at a low concentration (estimated value) of 10µg/mL (potency) and 10 % methanol.
(b) Standard of manufacturing method
The salinomycin producing strain of Streptomyces albus is aerobically cultured, after thecultivation solids are separated by filtration, and salinomycin in the solids are extracted withan organic solvent. This solution is concentrated, filtered and crystallized as sodium salt
under alkaline conditions. The obtained crystals are washed and dried.
(c) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is pieces, particles, powder or in which the raw material for manufacturingsalinomycin sodium (part 1) is mixed with carriers or granulated.
Potency: The potency test shows that 100 µg (potency) or less is contained in 1 mg, and thatit contains 85~125 % of the label potency.
Physical and chemical properties:
i. This product is pale yellow-white to pale brown small powder or particles with a
specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
iii. There is no mold in this product.

502

Provisional Translation from Japanese OriginalConfirmation test: According to the label potency of this product, the amount containing
approximately 30 mg (potency) of salinomycin is weighed, shaken with 10 mL of
methanol, and filtered. This solution 2 mL is measured, and the confirmation test ⅰ. forthe raw material for manufacturing of salinomycin sodium (part 1) is hereinafter appliedmutatis mutandis.
Loss on drying: 12.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing ofsalinomycin sodium (part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of salinomycin sodium(part 1) are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of salinomycin sodium (part 1) are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount required for testing is weighed to three significant digits and the value isrecorded. It is added with a constant volume of methanol using a volumetric pipette to
make its concentration approximately 400 µg/mL (potency), stirred or shaken, andfiltered or centrifuged. The resulting filtrate or supernatant is used as a sample stock
solution. The volume required for testing of the sample stock solution is measured by a
volumetric pipette, and the provisions of the raw material for manufacturing ofsalinomycin sodium (part 1) are hereinafter applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of salinomycin sodium (part 1) ismixed with carriers, and as appropriate particle-seized or sieved.
(c) Standard of storage method
The storage method of the raw materials for manufacturing of salinomycin sodium (part1) are applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture“使用上の注意 この飼料添加物は、鶏又は牛に過剰投与した場合発育障害がおこるので、定められた添加量を厳守するとともに、均一に配合するよう注意するこ
と。”

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Provisional Translation from Japanese Original

Precautions: Overdose of this feed additive causes developmental disorders in chicken or
cattle, so strictly follow the specified amount to be added and make sure to mix it uniformly.
Salinomycin sodium (part 2)
A. Raw material for manufacturing (Part 1)(a) Compositional standards
Potency: The potency test of this product shows that 300 μg (potency) or more is containedin 1 mg.
Physical and chemical properties: This product is pale brown powder with a specific odor.
Confirmation test:
i. 0.1 g (0.05~0.14 g) of this product is added with 10 mL of methanol, shaken well, andfiltered. 2 mL of the filtrate is added with 2 mL of vanillin-hydrochloric acid test
solution, shaken, and warmed for 5 minutes. The resulting solution is red.
ii. 30 mg (29.5~30.4 mg) of this product is added with 100 mL of methanol, shaken well,and filtered. 10 mL of the filtrate is added with 1 mL of 1 mol/L hydrochloric acid test
solution, shaken well, and allowed to stand at room temperature for 5 minutes. In themeasurement of the ultraviolet absorption spectrum of this solution, the absorption
maximum is at the wavelengths of 285~300 nm.
iii. 1 g (0.5~1.4 g) of this product is added with 10 mL of methanol, shaken well, andfiltered. The filtrate is evaporated to dryness on a water bath. The resulting residue
gives the qualitative reaction i. of sodium salt.
Purity test:
i. pH: The pH of a suspension of this product in water (1 → 100) shall be 8.0 to 10.0.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution using method No. 2 of the heavy metals test method. When heavy metal istested using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 μg/g or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared using method No. 3 of the arsenic test method. The arsenic test is performedin the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 μg/g or less)
Loss on drying: 7.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 45.0 % or less (1 g)
Nitrogen: 0.2~2.0 % (Kjeldahl method)
Crude fat: 47.0~85.0 %504

Provisional Translation from Japanese Original

Crude fiber: 2.0 % or lessPotency test:
Agar plate: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Preparation of working standard diluent: The provisions of the raw material for
manufacturing of Salinomycin sodium (part 1) are applied mutatis mutandis.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a certain
volume of methanol using a volumetric pipette to make its concentration (estimatedvalue) approximately 400 μg/mL (potency), stirred or shaken, and filtered or
centrifuged. The filtrate or supernatant is used as a sample stock solution. The volume
required for testing of the sample stock solution is measured using a volumetricpipette, and thereafter the provisions of the raw material for manufacturing ofSalinomycin sodium (part 1) are applied mutatis mutandis.
(b) Standard of manufacturing method
The Salinomycin producing strain of Streptomyces albus is aerobically cultured. After thecultivation, the solids are separated through filtration with adding a filter aid where
necessary. The solids are dried, added with silicic acid, light anhydrous silicic acid, or silicic
acid anhydride at a ratio within 12% of the mass of the dried solids, and crushed and mixed.
(c) Standard of storage method
It shall be stored in a sealed container.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“サリノマイシンナトリウム（飼料級）”
Salinomycin sodium (feed grade)
B. Raw material for manufacturing (Part 2)(a) Compositional standards
Potency: The potency test of this product shows that 200 μg (potency) or more is containedin 1 mg.
Physical and chemical properties: This product is pale brown powder or particles with a
specific odor.
Confirmation test:505

Provisional Translation from Japanese Originali. 0.1 g (0.05~0.14 g) of this product is added with 10 mL of methanol, shaken well, andfiltered. 2 mL of the filtrate is added with 2 mL of vanillin-hydrochloric acid test
solution, shaken, and warmed for 5 minutes. The resulting solution is red.
ii. 30 mg (29.5~30.4 mg) of this product is added with 100 mL of methanol, shaken well,and filtered. 10 mL of the filtrate is added with 1 mL of 1 mol/L hydrochloric acid test
solution, shaken well, and allowed to stand at room temperature for 5 minutes. In themeasurement of the ultraviolet absorption spectrum of this solution, the absorption
maximum is at the wavelengths of 285~300 nm.
Purity test:
i. pH: The pH of a suspension of this product in water (1 → 100) shall be 8.0 to 10.0.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution using method No. 2 of the heavy metals test method. When heavy metal istested using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 μg/g or less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared using method No. 3 of the arsenic test method. The arsenic test is performedin the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 μg/g or less)
Loss on drying: 7.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 60.0 % or less (1 g)
Nitrogen: 0.2~2.0 % (Kjeldahl method)
Crude fat: 30.0~40.0 %Crude fiber: 2.0 % or lessPotency test:
Agar plate: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Preparation of working standard diluent: The provisions of the raw material for
manufacturing of Salinomycin sodium (part 1) are applied mutatis mutandis.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a certain
volume of methanol using a volumetric pipette to make its concentration (estimatedvalue) approximately 400 μg/mL (potency), stirred or shaken, and filtered or
centrifuged. The filtrate or supernatant is used as a sample stock solution. The volume

506

Provisional Translation from Japanese Original

required for testing of the sample stock solution is measured using a volumetricpipette, and thereafter the provisions of the raw material for manufacturing ofSalinomycin sodium (part 1) are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the Salinomycin producing strain of Streptomyces albus is aerobicallycultured, and after the cultivation the culture solution is added with sodium carboxymethyl
cellulose and calcium carbonate, concentrated, added with light anhydrous silicic acid, anddried.
(c) Standard of storage method
It shall be stored in a sealed container.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“サリノマイシンナトリウム（飼料級）”
Salinomycin sodium (feed grade)
C. Preparation (Part 1)(a) Compositional standards
This product is pieces or powder in which fillers are mixed into the raw material for
manufacturing (Part 1) of Salinomycin sodium (part 2).
Potency: The potency test of this product shows that 100 μg (potency) or less is contained in
1 mg, and that it contains 85~125 % of potency on the label.
Physical and chemical properties:
i. This product is pale yellowish white to pale brown pieces or powder with a specificodor.
ii. This product passes through a standard 2.00 mm mesh sieve.
iii. No molding is observed in this product.
Confirmation test:i. According to the labeled potency of this product, the amount containing approximately
30 mg (potency) of Salinomycin is weighed, added with 10 mL of methanol, shaken,
and filtered. 2 mL of the filtrate is measured, and thereafter the confirmation test i. of
the raw material for manufacturing of Salinomycin sodium (part 2) is applied mutatis
mutandis.
ii. According to the labeled potency of this product, the amount containing approximately
10 mg (potency) of Salinomycin is weighed, added with 100 mL of methanol, shaken,
and filtered. 10 mL of the filtrate is measured, and thereafter the confirmation test ii. of507

Provisional Translation from Japanese Original

the raw material for manufacturing of Salinomycin sodium (part 2) is applied mutatis
mutandis.
iii. 1 g (0.5~1.4 g) of this product is weighed, added with 10 mL of methanol, shaken for
5 minutes, and filtered. The filtrate is pale yellow to yellow.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Preparation of working standard diluent: The provisions of the raw material for
manufacturing of Salinomycin sodium (part 1) are applied mutatis mutandis.
Preparation of sample solution: According to the labeled potency of this product, theamount required for testing is weighed to three significant digits and the value isrecorded. It is added with a certain volume of methanol using a volumetric pipette to
make its concentration approximately 400 μg/mL (potency), and thereafter theprovisions of the raw material for manufacturing of Salinomycin sodium (part 2) areapplied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing (Part 1) of Salinomycin sodium(part 2) is mixed with carriers.
(c) Standard of storage method
The storage method of the raw material for manufacturing (Part 1) of Salinomycin
sodium (part 2) is applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“サリノマイシンナトリウム（飼料級）”
Salinomycin sodium (feed grade)“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture“使用上の注意 この飼料添加物は、鶏に過剰投与した場合発育障害がおこるので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”
Precautions: Overdose of this feed additive causes developmental disorders in chicken, so
strictly follow the specified amount to be added and make sure to mix it uniformly.

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Provisional Translation from Japanese Original

D. Preparation (Part 2)(a) Compositional standards
This product is pieces, particles or powder in which fillers are mixed into the raw material
for manufacturing (Part 2) of Salinomycin sodium (part 2).
Potency: The potency test of this product shows that 200 μg (potency) or less is contained in
1 mg, and that it contains 85~125 % of potency on the label.
Physical and chemical properties:
i. This product is pale yellowish white to pale brown pieces, particles or powder with a
specific odor.
ii. This product passes through a standard 2.00 mm mesh sieve.
iii. No molding is observed in this product.
Confirmation test: The confirmation test of the preparation (Part 1) of Salinomycin sodium(part 2) is applied mutatis mutandis.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw material for manufacturing of Salinomycin sodium(part 1) are applied mutatis mutandis.
Preparation of working standard diluent: The provisions of the raw material for
manufacturing of Salinomycin sodium (part 1) are applied mutatis mutandis.
Preparation of sample solution: The provisions of the preparation (Part 1) of Salinomycin
sodium (part 2) are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing (Part 2) of Salinomycin sodium(part 2) is mixed with carriers.
(c) Standard of storage method
The storage method of the raw material for manufacturing (Part 2) of Salinomycin
sodium (part 2) is applied mutatis mutandis.
(d) Standards of the label
The standards of the label of the preparation (Part 1) of Salinomycin sodium (part 2) areapplied mutatis mutandis.

509

Provisional Translation from Japanese Original(118) Semduramicin Sodium
A. Raw material for manufacturing(a) Compositional standards
Potency: This product is sodium salt of semduramicin. The potency test shows that 860 µg(potency) or more is contained in 1 mg.
Physical and chemical properties:
i. This product is white to grayish white crystalline powder.
ⅱ. This product is easy to lightly dissolve in methanol, hard to lightly dissolve in ethanol,
hard to dissolves in dichloromethane and ether and hardly dissolves in water andisooctane.
Confirmation test:
i. This product and working reference standard are measured by the potassium bromide
disk method of the infrared absorption spectroscopy and their spectra are compared,
and then absorption is observed at approximately 3,450 cm−1, 2,972 cm−1, 2,935 cm−1,
1,595 cm−1, 1,460 cm−1, 1,381 cm−1, and 1,063 cm−1.
ⅱ. 5 mg (4.5~5.4 mg) of this product and working reference standard are each weighed,and both are dissolved with 5 mL of methanol to prepare a sample solution and astandard solution, respectively. 5 µL each of the sample and standard solutions are
spotted on a thin layer plate prepared using silica gel for thin-layer chromatography.
Next, they are developed to approximately 10 cm with developing solvent, a mixed
solution of ethyl acetate and glacial acetic acid (4:1), and the thin layer plate is air
dried. It is then sprayed with a vanillin-sulfuric acid-ethanol test solution and heated at
105 °C for approximately 10 minutes. Then, the main spots obtained from the sample
and standard solutions are red-brown and the Rf values of them are equal.
Purity test:i. Specific rotation: 0.25 g of this product is weighed to the digit of 0.001 g and the value
is recorded. It is dissolved with methanol to make 25 mL. The optical rotation of thissolution shall be [α]20
D = +19.0 ~ +23.0°
ⅱ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
iii. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by510

Provisional Translation from Japanese Original

the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
ⅳ. Hydroxysemduramicin: Approximately 25 mg of this product of is weighed anddissolved with 25 mL of a mixed solution of ethyl acetate, isooctane, glacial acetic
acid and triethylamine (375:125:2:1). This is used as a sample solution. When thesample solution of 50 µL is tested by the liquid chromatography under the following
conditions, the peak (the peak appearing when the time 1.5 fold longer than theretention time of the peak of semduramicin has passed) area of hydroxysemduramicin
shall be 2.5 % or less of the total area of the peaks obtained from the sample solution.
Operating condition:
Device: Consisting of a mobile phase delivery pump, coloring solution delivery pump,sample injector, column, reaction vessel, detector, and recorder. The column and thereaction vessel shall be maintained at constant temperature for use.
Detector: Ultraviolet absorptiometer (measurement wavelength: 522 nm)
Column: A stainless tube (internal diameter: 4.0 mm, length: about 250 mm) is filledwith 5 µm of silica gel.
Column temperature: Constant temperature around 25 °C
Reaction vessel (reaction coil): Tube with internal diameter of approximately 0.3 mm
and length of approximately 7.6 m
Mobile phase: Mixed solution of ethyl acetate, isooctane, glacial acetic acid andtriethylamine (375:125:2:1)
Coloring solution: A mixture 250 mL of absolute ethanol and 10 mL of sulfuric acid is
added in a solution of 15 g (14.5~15.4 g) of vanillin dissolved in 250 mL ofabsolute ethanol.
Mobile phase flow rate: 0.6 mL/min
Coloring solution flow rate: 0.3 mL/min
Column selection: A column to be used is selected when the solution, which isprepared by the dissolving of the working reference standard and sodiumhydroxysemduramicin in the mobile phase and adjusting its concentration to 0.02
mg/mL, is operated under the above conditions and both substances are completely
separated.
Detection sensitivity: The peak of semduramicin obtained 50 µL of the sample
solution (1 → 50) in the mobile phase is adjusted to 10~40 mm.
Area measurement range: Three times as much as the retention time of
semduramichin.
Loss on drying: 2.5 % or less (1 g, decompression, 100 °C, 3 hours)

511

Provisional Translation from Japanese Original

Ignition residue: 9.0 % or less (1 g)
Potency test:
Agar plate (single layer): 20 mL of buffer No. 19 mixed with the test strain is used (21mL in a Petri dish with internal diameter of 100 mm). It shall be a perforated agar
plate.
Test strain: Bacillus subtilis ATCC 6633 is used.
Preparation of working standard diluent: The amount of working standard diluent
required for testing is weighed to three significant digits and the value is recorded. It isdissolved with an adequate volume of methanol to make an accurate constant volume
at a concentration of approximately 1 mg/mL (potency). This is used as a diluted stock
solution. The volume of the diluted stock solution required for testing is measured
using a volumetric pipette, accurately diluted with water and methanol to prepare theworking standard diluent at a high concentration of 20 µg/mL (potency) with 25 %methanol and the working standard diluent at a low concentration of 5 µg/mL(potency) with 25 % methanol.
Preparation of the sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved withmethanol and is further added with methanol to make an accurate constant volume at aconcentration approximately 1 mg/mL (estimated value) (potency). This is used as asample stock solution. The volume of the sample stock solution required for testing ismeasured using a volumetric pipette and accurately diluted with water and methanol toprepare the sample solution at a high concentration of 20 µg/mL (potency) with 25 %methanol and the sample solution at a low concentration of 5 µg/mL (potency) with25 % methanol.
(b) Standard of manufacturing method
The semduramicin producing strain of Actinomadura roseorufa is aerobically cultured,
after the cultivation, semduramicin sodium is diluted from concentrated slurry of the culture
solution using an organic solvent under alkaline conditions. The crystals obtained from theextract are dried.
(c) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is pieces or powder of the mixture of the raw material for manufacturing ofsemduramicin sodium and carriers.

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Provisional Translation from Japanese Original

Potency: The potency test shows that 50 µg (potency) or less is contained in 1 mg, and thatit contains 85~125 % of the label potency.
Physical and chemical properties:
i. This product is grayish yellow to pale brown small pieces or powder.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test: According to the label potency of this product, the amount containing
approximately 50 mg (potency) of semduramycin sodium is weighed, added with 50 mL
methanol, vigorous stirred for 30 minutes and centrifuged. The supernatant is used as asample stock solution. The confirmation test ⅱ. of the raw materials for manufacturing ofsemduramycin sodium are hereinafter applied mutatis mutandis.
Loss on drying: 12.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing ofsemduramicin sodium are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of senduramicin
sodium are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of senduramicin sodium are applied mutatis mutandis.
Preparation of the sample solution: Where appropriate, this product is ground, according
to the label potency, the amount required for testing is weighed to three significant
digits and the value is recorded. It is added with a constant volume of methanol using a
volumetric pipette to make its concentration approximately 1 mg/mL (potency),
vigorous stirred for 20 minutes and centrifuged. The volume required for testing of thesample stock solution is measured using a volumetric pipette, and the provisions of the
raw material for manufacturing of monensin sodium are hereinafter applied mutatis
mutandis.
(b) Standard of manufacturing method
The raw material for manufacturing of semduramicin sodium is mixed with carriers.
(c) Standard of storage method
The storage method of the raw materials for manufacturing of semduramicin sodeium areapplied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”

513

Provisional Translation from Japanese Original

Valid period: 2 years from the month following the manufacture“使用上の注意 この飼料添加物は、鶏に過剰投与した場合発育障害がおこるので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”
Precautions: Overdose of this feed additive causes developmental disorders in chicken, so
strictly follow the specified amount to be added and make sure to mix it uniformly.

(119) Narasin
A. Raw material for manufacturing(a) Compositional standards
Potency: When this product is conducted potency test, it contains 100 µg (potency) or morein 1 mg.
Physical and chemical properties: This product is grayish brown to dark brown particles.
Confirmation test:
ⅰ. 0.8 g (0.75~0.84 g) of this product is weighed, added with 100 mL of methanol,
stirred for 20 minutes, and filtered. The filtrate is used as a sample solution.
Separately, the amount of working standard narasin containing approximately 10 mg(potency) of narasin is weighed and dissolved with 10 mL of methanol. This is used asa standard solution. 2 µL each of the sample and standard solutions are spotted on athin layer plate prepared using silica gel for thin-layer chromatography. Next, they aredeveloped to approximately 15 cm with developing solvent, a mixed solution of ethyl
acetate and strong ammonia solution (90:1), and the thin layer plate is air dried and isfurther dried at 105 °C for 10 minutes. It is uniformly sprayed with a vanillin-sulfuric
acid-ethanol coloring reagent and heated at 105 °C for approximately 10 minutes.
Then the main spots obtained from the sample and standard solutions are bluish purple
and the Rf values of them are equal.
ⅱ. 10 mg (9.5~10.4 mg) of this product is weighed, added with 50 mL of methanol (9 →10), stirred for 20 minutes, and filtered. This solution is further filtered using amembrane filter (0.45 µm) and the filtrate is used as a sample solution. Separately, 5
mg (4.5~5.4 mg) of working standard narasin is weighed and dissolved with 250 mLof methanol (9 → 10). This is used as a standard solution. 200 µL each of the sample
and standard solutions are tested by the liquid chromatography under the following
operating conditions, and the retention times of the peaks of narasin A obtained fromthe sample and standard solutions are consistent.
Operating condition:

514

Provisional Translation from Japanese Original

Device: Consisting of a mobile phase delivery pump, coloring solution delivery pump,sample injector, column, reaction vessel, detector, and recorder. The column and thereaction vessel shall be maintained at constant temperature for use.
Detector: Visible spectrophotometer (measurement wavelength: 520 nm)
Column: A stainless tube internal) diameter: 4.6 mm, length: about 250 mm) is filledwith 5 µm of octadecylsilylated silica gel for liquid chromatograph.
Column temperature: Constant temperature around 25 °C
Reaction vessel (reaction coil): Tube with internal diameter approximately 0.5 mm
and length of approximately 5.6 m
Mobile phase: Mixed solution of methanol, water and acetic acid (940:60:1)
Coloring solution: Mix 950 mL of methanol and 20 mL of acetic acid, dissolved 30 g
(29.5~30.4 g) of vanillin.
Mobile phase flow rate: 0.65 mL/min
Coloring solution flow rate: 0.65 mL/min
Column selection: 5 mg (4.5~5.4 mg) of each working standard monensin and narasin
is weighed, added with methanol (9 → 10) up to 250mL. When each 250mL of thissolutionis operated under the above operating conditions, elution is performedin the order of monensinA, narasinB. Using a solution having a degree of separationof 3.0 or more.
Area measurement range: Two times as much as the retention time of narasinA.
Purity test:ⅰ. Lead: 1.0 g (0.95~1.04 g) of this product is weighed, carbonized at approximately
200 °C for 2 hours, heated from 250 °C up to approximately 450 °C at the rate of
temperature increase of 50 °C/hour, and incinerated for two hours with thistemperature maintained. After cooling, it is added with 5 mL of nitric acid, covered
with a watch glass, and heated on a sand bath and then the watch glass is removed andthe product is almost dried. After cooling, it is added with approximately 25 mL ofhydrochloric acid (1 → 6) and gently heated for 10 minutes. After cooling, it istransferred to a 100 mL volumetric flask, added with water to the marked line to make100 mL, and filtered within 30 seconds. This is used as a sample solution. Separately,
1.0 mL of lead standard solution for atomic absorption is measured, transferred to a
100 mL volumetric flask, added with approximately 25 mL of hydrochloric acid (1 →6), and is further added with water to the marked line to make 100 mL. This is used asa standard solution. 30 mL each of the sample and standard solutions are measured,
added with 8 mL of phosphate, 2 mL of potassium iodide solution (6 → 10), and 10
mL of methyl isobutyl ketone using a volumetric pipette, vigorously shaken for515

Provisional Translation from Japanese Original

approximately 1 minute, and allowed to stand for a short time. Within 30 seconds,
away from direct sunlight, the methyl isobutyl ketone layers obtained from the sample
and standard solutions are tested by the atomic absorption spectrophotometry (flametype) at the wavelength of 283.3 nm, using a lead hollow cathode lamp as the lightsource, acetylene or hydrogen as a flammable gas, and air as a combustion-supportinggas. The absorbance of the methyl isobutyl ketone layer of the sample solution shall beequal to or lower than that of the standard solution (25 mg/kg or lower).
ⅱ. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (26 mg/kg or less).However, the volume of arsenic
standard solution for preparation of the standard color shall be 13 mL. The color iscorresponding to 0.026 mg of arsenic trioxide.
Arsenic standard solution: 20 mL of the arsenic standard solution is measured using
a volumetric pipette, transferred to a 1,000 mL volumetric flask, added with 5 mL of
dilute sulfuric acid, and then added with newly boiled and cooled water to the 1,000mL volumetric flask up to the marked line. 1 mL of this solution contains arsenic
trioxide 0.002 mg. This solution is prepared when needed and stored in a groundstopper bottle.
ⅲ. Content ratio of narasin A: When 200 µL each of the sample and standard solutions in
the confirmation test (2) are tested by the liquid chromatography, under the operating
conditions of the confirmation test (2), mutatis mutandis, the content ratio of narasin A
shall be 85 % or more.
Content ratio of narasin A in this product (%) = AT × 100
T

AT1: Peak area of nalasinA in sample solution
AT: Sum of peak areas of sample solution
Loss on drying: 10.0 % or less (1 g, 0.67kPa or less, 60 °C, 3 hours)
Nitrogen: 2.0 % or less (Kjeldahl method)
Crude fat: 27.0 % or less (2 g)
Crude fiber: 2.0 % or less (2 g)
Potency test:
Agar plate (single layer): 10 mL of the No. 15 medium mixed with the test strain isused (11 mL in a Petri dish with internal diameter of 100 mm).
Test strain: Bacillus subtilis ATCC 6633 is used.

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Provisional Translation from Japanese Original

Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is dissolved with sodium bicarbonate-methanol reagent, and is further addedwith sodium bicarbonate-methanol reagent to make a constant volume at a concentrationof approximately 1 mg/mL (potency) (estimated value). This is used as a diluted stock
solution. The volume required for testing of the diluted stock solution is measured using
a volumetric pipette, accurately diluted with water and methanol to prepare the working
standard diluent at a high concentration of 5 µg/mL (potency) (estimated value) with
30 % methanol and the working standard diluent at a low concentration of 1.25 µg/mL(potency) (estimated value) with 30 % methanol.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a constantvolume of methanol (9 → 10) using a volumetric pipette to make its concentration(estimated value) approximately 1 mg/mL (potency), stirred for 20 minutes and filtered.
The filtrate is used as a sample stock solution. The volume of the sample stock solution
required for testing is measured using a volumetric pipette, accurately diluted with waterand methanol to prepare the sample solution at a high concentration of 5 µg/mL(potency) (estimated value) with 30 % methanol and the sample solution at a low
concentration of 1.25 µg/mL (potency) (estimated value) with 30 % methanol.
(b) Standard of manufacturing method
For manufacturing, the narasin producing strain of Streptomyces aureofaciens isaerobically cultured, after the cultivation the culture solution is dried, added with bentonite
and calcium carbonate, and granulated.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
B. Preparation (Part 1)(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing ofnarasin and carriers.
Potency: When this product is conducted potency test, it contains 100 µg (potency) or lessin 1 mg including 85~125 % of the indicated potency.
Physical and chemical properties:
ⅰ. This product is grayish brown to brown or yellowish white to yellow powder orparticles.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.

517

Provisional Translation from Japanese OriginalConfirmation test:
ⅰ. 1.0 g (0.95~1.04 g) of this product is weighed, added with 100 mL of methanol,
stirred for 20 minutes, and filtered. The filtrate is used as a sample solution. The
confirmation test ⅰ. for the raw material for manufacturing of Narasin is appliedmutatis mutandis.
ⅱ. The confirmation test ⅱ. for the raw material for manufacturing of Narasin is appliedmutatis mutandis.
Loss on drying: 12.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Potency test: The provisions of the raw materials for manufacturing of narasin are appliedmutatis mutandis.
Agar plate (single layer): The provisions of the raw materials for manufacturing ofnarasin are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of narasin are appliedmutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of narasin are applied mutatis mutandis.
Preparation of sample solution: According to the label potency, the amount of theworking reference standard required for testing is weighed to three significant digits
and the value is recorded. It is added with a constant volume of a mixed solution of
methanol (9 → 10), using a volumetric pipette to make its concentration approximately
1 mg/mL (potency) (estimated value), stirred for 20 minutes and filtered. The filtrate isused as a sample stock solution. The volume of the sample stock solution required fortesting is measured using a volumetric pipette, then the provisions of the raw materials
for manufacturing of narasin are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of narasin is added with carriers.
(c) Standard of storage method
The standard storage method of the raw materials for manufacturing of narasin are
applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture518

Provisional Translation from Japanese Original“使用上の注意 この飼料添加物は、鶏に過剰投与した場合発育障害がおこる
ので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”Precautions: Overdose of this feed additive causes developmental disorders in chicken, so
strictly follow the specified amount to be added and make sure to mix it uniformly.

(120) Nosiheptide
Nosiheptide (part 1)
A. Raw material for manufacturing(a) Compositional standards
Potency: The potency test shows that 800 µg (potency) or more is contained in 1 mg.
Physical and chemical properties:
ⅰ. This product is pale yellow-gray-white to greenish pale yellow-brown crystals or
powder without odor or with a slightly specific odor.
ⅱ. This product is easy to slightly dissolve in cyclohexanone, hard to lightly dissolve intetrahydrofuran, hard to dissolve in acetone and chloroform, is extremely hard todissolve in ethanol, and hardly dissolves in water.
Confirmation test:
ⅰ. 50 mg (49.5~50.4 mg) of this product is weighed, added with 50 mL oftetrahydrofuran, used as a sample solution. Measure 1 mL of this solution, added with
2 mL of a mixed solution of methanol and ammonia solution (87:13) to 20 mL. In themeasurement of the absorption spectrum of this solution, the absorption maximum is at
the wavelengths of 405~411 nm.
ⅱ. 10 mg (9.5~10.4 mg) of this product is measured, added with 0.1 g (0.05~0.14 g) of
sodium hydroxide, gradually heated to melt. After cooling, it is dissolved with 1 mL of
distilled water, and added with 0.3 g (0.25~0.34 g) of zinc powder and 5 mL ofhydrochloric acid (1 → 2), and the produced gas discolors wet lead acetate paper toblack.
Purity test:
ⅰ. pH: The pH of solution (1 → 1000) of this product shall be 4.5~8.0.
ⅱ. Lead: 2.0 g (1.95~2.04 g) of this product is weighed and heated at 500 °C for 4 hours.
After cooling, it is added with 5 mL each of nitric acid and perchloric acid, covered
with a watch glass, and heated on a sand bath to transparent, and the watch glass isremoved to make it almost dry. After cooling, it is added with approximately 10 mL of
dilute nitric acid (1 → 3) and gently heated for 10 minutes. After cooling, it is519

Provisional Translation from Japanese Originaltransferred to a 20 mL volumetric flask and added with dilute nitric acid (1 → 3) to themarked line to make 20 mL. This is used as a sample solution. Separately, 2.0 mL oflead standard solution for atomic absorption is measured, transferred to a 50 mL
volumetric flask, and added with dilute nitric acid (1 → 3) to the marked line to make
50 mL. This is used as a standard solution. When the sample and standard solutions aretested by atomic absorption spectrophotometry (flame type) at the wavelength of 283.3
nm, using a lead hollow cathode lamp as the light source, acetylene or hydrogen as aflammable gas, and air as a combustion-supporting gas, the absorbance of the sample
solution shall be equal to or lower than that of the standard solution (10 mg/kg or
lower).
ⅲ. Copper: The sample solution obtained in ⅱ. 5 mL is measured by a volumetric pipette,
transferred to a 50 mL volumetric flask, and added with dilute nitric acid (1 → 3) tothe marked line to make 50 mL. This is used as a sample solution. Separately, 2.0 mLof copper standard solution is measured, transferred to a 50 mL volumetric flask andadded with dilute nitric acid (1 → 3) to the marked line to make 50 mL. This is used asa standard solution. When the sample and standard solutions are tested by the atomic
absorption spectrophotometry (flame type) at the wavelength of 324.7 nm, using acopper hollow cathode lamp as the light source, acetylene or hydrogen as a flammablegas, and air as a combustion-supporting gas, the absorbance of the sample solution
shall be equal to or lower than that of the standard solution (100 mg/kg or lower).
ⅳ. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: 7.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 5.0 % or less (1 g)
Potency test:
Agar plate (single layer): 10 mL of the No. 7 medium mixed with the test strain is used
(11 mL in a Petri dish with internal diameter of 100 mm).
Test strain: Micrococcus luteus ATCC 9341 is used.
Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is dissolved with dimethylformamide, and is further added withdimethylformamide to make a constant volume at a concentration of approximately 1
mg/mL (potency) (estimated value). This is used as a diluted stock solution. The
volume required for testing of the diluted stock solution is measured using a520

Provisional Translation from Japanese Original

volumetric pipette, accurately diluted with a mixed solution of buffer No. 4 and etanol(85:15) to prepare the working standard diluent at a high concentration of 2 µg/mL(potency) (estimated value) and the working standard diluent at a low concentration of
0.5 µg/mL (potency) (estimated value).
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved withdimethylformamide and is further added with dimethylformamide to make an accurate
constant volume at a concentration of approximately 1 mg/mL (potency) (estimatedvalue). This is used as a sample stock solution. The volume of the sample stock
solution required for testing is measured using a volumetric pipette and accurately
diluted with a mixed solution of buffer No. 4 and ethanol (85:15) to prepare the sample
solution at a high concentration of 2 µg/mL (potency) (estimated value) and thesample solution at a low concentration of 0.5 µg/mL (potency) (estimated value).
(b) Standard of manufacturing method
For manufacturing, the nosiheptide producing strain of Streptomyces actuosus isaerobically cultured, after the cultivation, solids are separated by filtration. Nosiheptide in
the solids is extracted with an organic solvent, the extract is concentrated, the concentrate isadded with isopropanol, and the resulting precipitation is washed with isopropanol once or
twice and then dried.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
B. Preparation(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing of
nosiheptide (part 1) and carriers.
Potency: When this product is conducted potency test, it contains 85~125 % of the indicatedpotency.
Physical and chemical properties:
ⅰ. This product is pale yellow to light brown powder or particles without odor, or with aslightly specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:ⅰ. According to the label potency of this product, the amount containing approximately
50 mg (potency) of nosiheptide is weighed, added 50 mL of tetrahydrofuran, hardly521

Provisional Translation from Japanese Original

shaken for 20 minutes, and filtered. The confirmation test i. for the raw material for
manufacturing of nosiheptide (part 1) is hereinafter applied mutatis mutandis.
ⅱ. According to the label potency of this product, the amount containing approximately
20 mg (potency) of nosiheptide is weighed, added 50 mL of tetrahydrofuran, hardly
shakenfor 20 minutes, and filtered. Measure 10 mL of this solution, evaporated todryness, the confirmation test ii. for the raw material for manufacturing of nosiheptide(part 1) is hereinafter applied mutatis mutandis.
Loss on drying: 15.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing of
nosiheptide (part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of nosiheptide (part 1)
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of nosiheptide (part 1) are applied mutatis mutandis.
Preparation of sample solution: According to the label potency, the amount of theworking reference standard required for testing is weighed to three significant digits
and the value is recorded. It is added with a constant volume of a mixed solution ofdimethylformamide, using a volumetric pipette to make its concentration
approximately 1 mg/mL (potency), vigorously stirred for 20 minutes and filtered or
centrifuged. The filtrate is used as a sample stock solution. The volume of the sample
stock solution required for testing is measured using a volumetric pipette, then theprovisions of the raw materials for manufacturing of nosiheptide (part 1) are appliedmutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of nosiheptide is added withcarriers.
(c) Standard of storage method
The standard storage method of the raw materials for manufacturing of nosiheptide areapplied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Validity period: 2 years from the month following production522

Provisional Translation from Japanese Original

Nosiheptide (part 2)
A. Raw material for manufacturing(a) Compositional standards
Potency: The potency test shows that 65 µg (potency) or more is contained in 1 mg.
Physical and chemical properties: This product is yellowish-brown to dark brown powder orparticle with a specific odor.
Confirmation test:
ⅰ. 0.5 g (0.45~0.54 g) of this product is weighed, added with 50 mL of a mixed solutionof tetrahydrofuran and water (80:20), vigorously shaken for 20 minutes, and filtered.
This filtrate is used as a sample solution. 1 mL of this sample solution is measured andadded with a mixed solution of methanol and ammonia (87:13) to make 20 mL. In themeasurement of the absorption spectrum, the absorption maximum of this solution is at
the wavelengths of 405~411 nm.
ⅱ. 0.1 g (0.05~0.14 g) of this product is measured, added with 0.1 g (0.05~0.14 g) of
sodium hydroxide, gradually heated to melt. After cooling, it is dissolved with 1 mL of
distilled water, and added with 0.3 g (0.25~0.34 g) of zinc powder and 5 mL ofhydrochloric acid (1 → 2), and the produced gas discolors wet lead acetate paper toblack.
Purity test:
ⅰ. pH: The pH of solution (1 → 20) of this product shall be 4.5~8.0.
ⅱ. Lead: 2.0 g (1.95~2.04 g) of this product is weighed and heated at 500 °C for 4 hours.
After cooling, it is added with 5 mL of nitric acid and 5 mL of perchloric acid, covered
with a watch glass, and heated on a sand bath to transparent, and removed the watch
glass to make it almost dry. After cooling, it is added with appropriately 10 mL of
dilute nitric acid (1 → 3) and gently heated for 10 minutes. After cooling, it istransferred to a 20 mL volumetric flask and added with dilute nitric acid (1 → 3) to the
20 mL volumetric flask up to the marked line. This is used as a sample solution.
Separately, 1.0 mL of lead standard solution for atomic absorption is measured,transferred to a 50 mL volumetric flask, and added with dilute nitric acid (1 → 3) tothe 50 mL volumetric flask up to the marked line. This is used as a standard solution.
When the sample and standard solutions are tested by the atomic absorption
spectrophotometry (flame type) at the wavelength of 283.3 nm, using a lead hollow
cathode lamp as the light source, acetylene or hydrogen as a flammable gas, and air as
a combustion-supporting gas, the absorbance of the sample solution shall be equal toor lower than that of the standard solution (5 mg/kg or lower).

523

Provisional Translation from Japanese Originalⅲ. Copper: The sample solution obtained in ⅱ. 5 mL is measured by a volumetric pipette,
transferred to a 50 mL volumetric flask, and added with dilute nitric acid (1 → 3) tothe 50 mL volumetric flask up to the marked line. This is used as a sample solution.
Separately, 2.0 mL of copper standard solution is measured, transferred to a 50 mL
volumetric flask and added with dilute nitric acid (1 → 3) to the 50 mL volumetricflask up to the marked line. This is used as a standard solution. When the sample andstandard solutions are tested by the atomic absorption spectrophotometry (flame type)at the wavelength of 324.7 nm, using a copper hollow cathode lamp as the light source,
acetylene or hydrogen as a flammable gas, and air as a combustion-supporting gas, theabsorbance of the sample solution shall be equal to or lower than that of the standard
solution (10 mg/kg or lower).
ⅳ. Arsenic: 1.0 g (0.95~1.04 g) of this product is transferred to a dissolution flask, addedwith 10 mL of nitric acid and 5 mL of sulfuric acid, and gently heated. If the solution
remains brown, it is allowed to cool, is added with 1~2 mL of nitric acid, and heated.
This procedure is repeated until the solution is colorless to pale yellow. After cooling,
it is added with 0.5 mL of perchloric acid and heated until white smoke emerges. Aftercooling, it is added with 15 mL of saturated ammonium oxalate and heated until whitesmoke emerges. After cooling, it is added with water to make approximately 10 mL
and this is used as a sample solution. When the sample solution is tested for arsenic
using device A, the color of the absorbing solution shall not be darker than standard
color (2 mg/kg or lower).
Loss on drying: 13.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 20.0 % or less (0.5 g)
Nitrogen: 8.0 % or less (Kjeldahl method)
Crude fat: 22.0 % or lessCrude fiber: 6.0 % or lessPotency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing of
nosiheptide (part 1) are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of nosiheptide (part1)
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of nosiheptide (part 1) are applied mutatis mutandis.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved with amixed solution of dimethylformamide and buffer No. 4 (80:20) and is further added524

Provisional Translation from Japanese Original

with a mixed solution of dimethylformamide and buffer No. 4 (80:20) to make anaccurate constant volume at a concentration of approximately 1 mg/mL (potency)(estimated value), is vigorously shaken for 20 minutes, and filtered or centrifuged.
This is used as a sample stock solution. The volume of the sample stock solution
required for testing is measured using a volumetric pipette. The provisions of the raw
material for manufacturing of nosiheptide (part 1) are hereinafter applied mutatis
mutandis.
(b) Standard of manufacturing method
For manufactuaring, the nosiheptide producing strain of Streptomyces actuosus isaerobically cultured, and after the cultivation, dried and crushed.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“ノシヘプタイド（飼料級）”
Nosiheptide (feed grade)
B. Preparation(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing of
nosiheptide (part 2) and carriers.
Potency: When this product is conducted potency test, it contains 85~125 % of the indicatedpotency.
Physical and chemical properties:
ⅰ. This product is light brown to brown powder or particles with a slightly specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:ⅰ. According to the label potency of this product, the amount containing approximately
50 mg (potency) of nosiheptide is weighed, and the confirmation test i. for the raw
material for manufacturing of nosiheptide (part 2) is hereinafter applied mutatis
mutandis.
ⅱ. According to the label potency of this product, the amount containing approximately 20
mg (potency) of nosiheptide is weighed, added 20 mL of a mixed solution oftetrahydrofuran and water (80:20), vigorously shaken for 20 minutes, and filtered. The
filterer is used as a sample solution. Measure 10mL of this solution, evaporated to525

Provisional Translation from Japanese Original

dryness, the confirmation test ⅱ. for the raw material for manufacturing of nosiheptide(part 2) is hereinafter applied mutatis mutandis.
Loss on drying: 15.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing of
nosiheptide (part 2) are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of nosiheptide (part 2)
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of nosiheptide (part 2) are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount required for testing is measured to three significant digits and the value isrecorded. It is added with a constant volume of a mixed solution ofdimethylformamide and buffer No. 4 (80:20) using a volumetric pipette to make its
concentration approximately 1 mg/mL (potency), vigorously shaken for 20 minutes,
and filtered or centrifuged. This is used as a sample stock solution. The volume of thesample stock solution required for testing is measured using a volumetric pipette. The
provisions of the raw material for manufacturing of nosiheptide (part 2) are hereinafter
applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of nosiheptide (part2) is addedwith carriers.
(c) Standard of storage method
The standard storage method of the raw materials for manufacturing of nosiheptide (part2) are applied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“ノシヘプタイド（飼料級）”
Nosiheptide (feed grade)“有効期間 製造の翌月から２年”
Validity period: 2 years from the month following production

526

Provisional Translation from Japanese Original(121) Bicozamycin
A. Raw material for manufacturing(a) Compositional standards
Potency: When this product is conducted potency test, it contains 850µg (potency) or morein 1mg.
Physical and chemical properties:
ⅰ. This product is white to brownish white crystals or powder without odor or with aslightly specific odor.
ⅱ. This product is easy to dissolve in water, slightly easy to dissolve in methanol, hard todissolve in acetone or etanol, and hardly dissolves in ether or chloroform.
Confirmation test:
ⅰ. This product in the solution (1 → 100) 10 mL is added with 1 mL of ninhydrin reagent
and gently shaken in a water bath for 15 minutes. The resulting solution is purple.
ⅱ. The solution of this product (1 → 10) of 10 mL is gently added with anthrone test
solution, and the color of interface is yellow-brown.
Purity test:
ⅰ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ⅱ. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (2 mg/kg or less).
Loss on drying: 6.0 % or less (1 g, reduced pressures, 60 °C, 3 hours)
Ignition residue: 0.5 % or less (1 g)
Potency test:
Agar plate: Medium No. 14 is used for both the media of base and inoculated layers.
Test strain: Escherichia coli ATCC 27166 is used.
Preparation of working standard diluent: The amount of the working reference standard
required for testing is measured to three significant digits and the value is recorded. It
is dissolved with buffer No. 3 and is further added with buffer No. 3 to make anaccurate constant volume at a concentration of appropriately 1 mg/mL (potency). Thisis used as a diluted stock solution. The volume required for testing of the diluted stock
solution is measured using a volumetric pipette and accurately diluted with buffer No.

527

Provisional Translation from Japanese Original

3 to prepare the working standard diluent at a high concentration of 120 µg/mL(potency) and the working standard diluent at a low concentration of 30 µg/mL(potency).
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved with buffer
No. 3 and is further added with buffer No. 3 to make an accurate constant volume at aconcentration of approximately 1 mg/mL (potency) (estimated value). This is used as asample stock solution. The volume of the sample stock solution required for testing ismeasured using a volumetric pipette, accurately diluted with buffer No. 3 to prepare
the sample solution at a high concentration of 120 µg/mL (potency) (estimated value)
and the sample solution at a low concentration of 30 µg/mL (potency) (estimatedvalue).
(b) Standard of manufacturing method
For manufacturing, the bicozamycin producing strain of Streptomyces griseoflavus isaerobically cultured, after the cultivation the bacterial cells are removed. Next, bicozamycinin this culture solution is adsorbed on a resin, eluted using a hydrous organic solvent, andthe eluate is concentrated to crystallize, or the culture filtrate is purified using ion exchange
resin, the purified solution is concentrated to crystallize.
(c) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is powder or particles of mixture of the raw material for manufacturing of
bicozamycin and carriers.
Potency: When this product is conducted potency test, it contains 85~125 % of the indicatedpotency.
Physical and chemical properties:
ⅰ. This product is white to light-brown powder or particles without odor, or with a slightly
specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:ⅰ. According to the label potency of this product, the volume containing approximately
100 mg (potency) of bicozamycin is weighed, added with 40 mL of a mixed solution ofchloroform and methanol (2:1), shaken well for 30 minutes, and filtered. The insoluble
matter on the filter is washed twice with 5 mL of a mixed solution of chloroform and528

Provisional Translation from Japanese Original

methanol (2:1) each, the filtrate and the washings are mixed, and the solvent is distilled
off under reduced pressure. The residue is added with 10 mL of water and 5 mL ofchloroform, shaken well for 1 minute, and if necessary centrifuged. The aqueous layer
is used as a sample solution. This sample solution of 5 mL is measured, added with 0.5
mL of ninhydrin test solution, heated in a water bath for 15 minutes, and then the
resulting solution is purple.
ⅱ. The amount of the working standard bicozamycin containing approximately 50 mg(potency) of bicozamycin is weighed and dissolved with 5 mL of water. This is used asa standard solution. 5 µg each of the sample and standard solutions of (ⅰ) are spottedon a thin layer plate prepared using silica gel for thin-layer chromatography. Next,
they are developed to approximately 10 cm with the developing solvent, a mixed
solution of ethyl acetate, methyl ethyl ketone, formic acid and water (5:3:1:1), and thethin layer plate is air dried. It is heated at 80 °C for 30 minutes, allowed to cool, andevenly sprayed with alkaline blue tetrazolium test solution, the spots obtained from thesample and standard solutions are purple, and the Rf values of them are the equal.
Loss on drying: 15.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate (single layer): The provisions of the raw materials for manufacturing of
bicozamycin are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of bicozamycin are
applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of bicozamycin are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount required for testing is weighed to three significant digits and the value isrecorded. It is added with the adequate amount of chloroform, added with a constantvolume of buffer No. 3 using a volumetric pipette to make its concentration 1~10
mg/mL (potency), vigorously shaken, and if necessary filtered or centrifuged. The
aqueous layer is used as a sample stock solution. The volume required for testing of
the sample stock solution is measured using a volumetric pipette, and the provisions of
the raw material for manufacturing of bicozamycin are hereinafter applied mutatis
mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of bicozamycin is added withcarriers.

529

Provisional Translation from Japanese Original(c) Standard of storage method
It shall be stored in a sealed container.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Validity period: 2 years from the month following production(122) Flavophospholipol
A. Raw material for manufacturing (Part 1)(a) Compositional standards
Potency: When this product is conducted potency test, it contains 40 µg (potency) or morein 1 mg.
Physical and chemical properties: This product is brown to dark brown powder with specificodor.
Confirmation test: 0.1 g (0.05~0.14 g) of this product is weighed, added with 1 mL of
methanol (1 → 2), shaken well, and centrifuged. The supernatant is used as a sample
solution. Separately, 10 mL (9.5~10.4 mg) of the working standard flavophospholipol isweighed and dissolved with 5 mL of methanol. This is used as a standard solution. 0.1
mL each of the sample and standard solutions are spotted on a thin layer plate preparedusing silica gel with a fluorescent agent for thin-layer chromatography. Next, they aredeveloped with the developing solvent, a mixed solution of isopropanol and strong
ammonia solution (65:35), for 4 hours and the thin layer plate is air dried. When it isirradiated with ultraviolet light (dominant wavelength: 254 nm), the Rf values of spots ofthe sample and standard solutions are equal.
Purity test:
ⅰ. pH: The pH of solution (1 → 1000) of this product shall be 4.5~8.0.
ⅱ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (30 mg/kg or less).
ⅲ. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by530

Provisional Translation from Japanese Original

the method using device A. The color of the absorbing solution shall not be darker
than the standard color (5 mg/kg or less).
Loss on drying: 4.0 % or less (1 g, 0.67 kPa or less, 3 hours)
Ignition residue: 20.0 % or less (1 g)
Nitrogen: 3.5~4.5 % (Kjeldahl method)
Crude fat: 22.0 % or lessCrude fiber: 4.0 % or lessPotency test:
Agar plate: Medium No. 12 is used for both the media of base and inoculated layers.
However, the medium of inoculated layer is dissolved and mixed with 1 mg of
methylene blue and 40 mg of boric acid per 100 mL of medium.
Test strain: Bacillus cereus ATCC 19637 is used.
Preparation of working standard diluent: The amount of the working reference standard
required for testing is weighed to three significant digits and the value is recorded. It isdissolved with methanol (1 → 2), and is further added with methanol (1 → 2) to makea constant volume at a concentration of approximately 1 mg/mL (potency). This isused as a diluted stock solution. The volume required for testing of the diluted stock
solution is measured using a volumetric pipette. After diluting this solution 10 times
with methanol (1 → 2), the volume of this solution is measured using a volumetricpipette, accurately diluted with buffer No.7 to prepare the working standard diluent at
a high concentration and at a low concentration.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a constantvolume of methanol (1 → 2) using a volumetric pipette to make its concentration(estimated value) approximately 100 µg/mL (potency), and stirred well. It is adjusted
to pH 8.0 with 1 mol/L sodium hydroxide solution, attached a reflux condenser, andheated at 85 °C for 15 minutes. After cooling, it is adjusted to pH 7.0 withhydrochloric acid solution. It is added small amount of methanol (1 → 2) to makeexactly the constant volume using a volumetric flask. The supernatant solution is usedas the sample stock solution.The volume of the sample stock solution required fortesting is measured using a volumetric pipette, accurately diluted with buffer No.7 toprepare the sample solution at a high concentration of 6 µg/mL (potency) (estimatedvalue) and the sample solution at a low concentration of 1.5 µg/mL (potency)(estimated value).

531

Provisional Translation from Japanese Original(b) Standard of manufacturing method
For manufacturing, the flavophospholipol producing strain of Streptomyces
bambergiensis, Streptomyces ghanaensis, Streptomyces geyseriensis or Streptomycesederensis is aerobically cultured, after the cultivation, the culture solution is dried(c) Standard of storage method
It shall be stored in a light resistant sealed container.
B. Preparation (Part 2)(a) Compositional standards
Potency: When this product is conducted potency test, it contains 100 µg (potency) or morein 1 mg.
Physical and chemical properties: This product is brown to dark brown powder with specificodor.
Confirmation test: 0.1 g (0.05~0.14 g) of this product is weighed, added with 1 mL of
methanol (1 → 2), shaken well, and centrifuged. The supernatant is used as a sample
solution. Separately, 10 mL (9.5~10.4 mg) of the working standard flavophospholipol isweighed and dissolved with 5 mL of methanol. This is used as a standard solution. 0.1
mL each of the sample and standard solutions are spotted on a thin layer plate preparedusing silica gel with a fluorescent agent for thin-layer chromatography. Next, they aredeveloped with the developing solvent, a mixed solution of isopropanol and strong
ammonia solution (65:35), for 4 hours or more and the thin layer plate is air dried. When
it is irradiated with ultraviolet light (dominant wavelength: 254 nm), the Rf values ofspots of the sample and standard solutions are equal.
Purity test:
ⅰ. pH: The pH of solution (1 → 50) of this product shall be 8.0~9.5.
ⅱ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 3.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (30 mg/kg or less).
ⅲ. Arsenic: 0.40 g (0.395~0.404 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (5 mg/kg or less).
Loss on drying: 10.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 40.0 % or less (1 g)
Nitrogen: 2.5~4.5 % (Kjeldahl method)532

Provisional Translation from Japanese Original

Crude fat: 22.0 % or lessCrude fiber: 4.0 % or lessPotency test:
Agar plate: Medium No. 12 is used for both the media of base and inoculated layers.
However, the medium of inoculated layer is dissolved and mixed with 1 mg of
methylene blue and 40 mg of boric acid per 100 mL of medium.
Test strain: Bacillus cereus ATCC 19637 is used.
Preparation of working standard diluent: The amount of the working reference standard
required for testing is weighed to three significant digits and the value is recorded. It isdissolved with methanol (1 → 2), and is further added with methanol (1 → 2) to makea constant volume at a concentration of approximately 1 mg/mL (potency). This isused as a diluted stock solution. The volume required for testing of the diluted stock
solution is measured using a volumetric pipette. After diluting this solution 10 times
with methanol (1 → 2), the volume of this solution is measured using a volumetricpipette, accurately diluted with buffer No.7 to prepare the working standard diluent at
a high concentration of 6 µg/mL (potency) and the working standard diluent at a low
concentration of 1.5 µg/mL (potency).
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a constantvolume of methanol (1 → 2) using a volumetric pipette to make its concentration(estimated value) approximately 100 µg/mL (potency), and stirred well. It is adjusted
to pH 8.0 with 1 mol/L hydrochloric acid solution, attached a reflux condenser, andheated at 85 °C for 15 minutes. After cooling, it is adjusted to pH 7.0 withhydrochloric acid solution. It is added small amount of methanol (1 → 2) to makeexactly the constant volume using a volumetric flask. The supernatant solution is usedas the sample stock solution.The volume of the sample stock solution required fortesting is measured using a volumetric pipette, accurately diluted with buffer No.7 toprepare the sample solution at a high concentration of 6 µg/mL (potency) (estimatedvalue) and the sample solution at a low concentration of 1.5 µg/mL (potency)(estimated value).
(b) Standard of manufacturing method
For manufacturing, the flavophospholipol producing strain of Streptomyces ghanaensis isaerobically cultured, after the cultivation the culture solution is concentrated, added withlight anhydrous silicic acid, and dried.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.

533

Provisional Translation from Japanese Original

C. Preparation(a) Compositional standards
This product is powder of mixture of the raw material for manufacturing offlavophospholipol and carriers.
Potency: When this product is conducted potency test, it contains 85~125 % of the indicatedpotency.
Physical and chemical properties:
ⅰ. This product is brown powder with a slightly specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test: According to the label potency of this product, the volume containing
approximately 5 mg (potency) of flavophospholipol is weighed, added with 1 mL of
methanol (1 → 2), and the provisions of the raw material for manufacturing of
bicozamycin are hereinafter applied mutatis mutandis.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate: The provisions of the raw materials for manufacturing of flavophospholipol
are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of flavophospholipol
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of flavophospholipol are applied mutatis mutandis.
Preparation of sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is added with a constantvolume of methanol (1 → 2) to make its concentration (estimated value)
approximately 100 µg/mL (potency), and stirred well. The provisions of the raw
materials for manufacturing of flavophospholipol are applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of flavophospholipol is mixed
with carriers, and as appropriate particle-seized and sieved.
(c) Standard of storage method
It shall be stored in a light resistant sealed container.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”

534

Provisional Translation from Japanese Original

Validity period: 2 years from the month following production(123) Monensin Sodium
A. Raw material for manufacturing(a) Compositional standards
Potency: This product is sodium salt of monensin. The potency test shows that 800 µg(potency) or more is contained in 1 mg.
Physical and chemical properties:
ⅰ. This product is slightly pale brown-white to pale orange-yellow powder or crystalline
powder with a slightly specific odor.
ⅱ. This product is easy to dissolve in ethanol and chloroform, hard to lightly dissolve in
acetone and hardly dissolves in water.
Confirmation test:
ⅰ. This product in methanol solution (1 → 1,000) 5 mL is added with 5 mL of vanillinhydrochloric acid test solution and gently shaken. The resulting solution is red.
ⅱ. This product in methanol solution (1 → 1,000) 5 mL is added with 5 mL of pdimethylaminobenzaldehyde-ethanol-sulfuric acid test solution and heated in water
bath at 70 ℃ for 5 minutes. The resulting solution is blue.
ⅲ. This product 3 g (2.5~3.4 g) is added with 20 mL of methanol and 2 mL of dilutedhydrochloric acid, gently boiled in water bath for 5 minutes, and added with 3 mL ofwater, cooled and filtered. After almost evaporating the filtrate, added with a small
amount of ethanol, and evaporate to dryness. A solution obtained by adding 10 mL ofwater to the residue and dissolving it shows a qualitative reaction of sodium salt.
Purity test:
ⅰ. pH: 0.1 g (0.05~0.14 g) of this product is weighed, added with 100 mL of methanol (9→ 10). The pH of this solution shall be 6.5~9.5.
ⅱ. Potency ratio of monensin A: According to the results of the potency test, the amountof this product containing approximately 50 mg (potency) of monensin is weighed tothree significant digits and the value is recorded. It is dissolved with methanol, and isfurther added with methanol to make an accurate constant volume at a concentration of
approximately 1 mg/mL (potency). This is used as a sample stock solution. The
volume required for testing of the sample stock solution using a volumetric pipette andadded with methanol to an accurate constant volume to prepare the sample solution ata high concentration of approximately 100 µg/mL (potency) and the sample solution ata low concentration of approximately 10 µg/mL (potency). Separately, in the same535

Provisional Translation from Japanese Originalmanner, working standard monensin is weighed to prepare the solutions of 100 µg/mL(potency), 10 µg/mL (potency), 5 µg/mL (potency), and 2.5 µg/mL (potency). They
are used as standard solutions S1, S2, S3, and S4.
The base line is set at the height of approximately 3 cm from the lower end of a layer
plate prepared using silica gel for thin-layer chromatography. The sample solution at ahigh concentration is spotted on the base line approximately 3 cm or more away fromthe edge, the sample solution at low concentration is spotted on the place 2.5 cm or
more away from the place of the high concentration solution, and 20 µg each of thestandard solutions are spotted at 2 cm or more intervals, and they are air dried. Next,
they are developed using ethyl acetate as the developing solvent at room temperature.
When the tip of the developing solvent reaches approximately 15 cm from the baseline, the thin layer plate is taken out and air dried, and it is developed and air dried
well again in the same way.
The surface of this thin layer plate is uniformly sprayed with the medium dissolvedin advance by heating in a water bath. Within 30 seconds, the thin layer plate agar side
up is put in a culture box, gently added with an adequate volume of the medium with
spore fluid maintained at 50~60 °C, and then uniformly spread. Then, it is solidified in
the horizontal culture box at room temperature, and cultured at 35~37 °C for 16~18
hours. The Rf values of the maximum inhibition zone (monensin A) and the second
maximum inhibition zone (monensin B) obtained from the sample solution are equal
to those obtained from the standard solution, and shall be 90 % or over when thepotency ratio of monensin A is determined.
Medium: 2.5 g (2.45~2.54 g) of yeast extract, 10 g (9.5~10.4 g) of glucose, 10 g
(9.5~10.4 g) of magnesium chloride, and 15 g (14.5~15.4 g) of agar are weighed,added with water to make 1,000 mL, and sterilized (pH 4.8~5.2).
Preparation of spore fluid: Bacillus subtilis ATCC 6633 is used as a test strain. The spore
fluid is prepared by the potency test specified in General Tests.
Measurement: As for monensin A in the standard solution S1, S2, S3, and S4, monensin A
in the sample solution at a low concentration, monensin B in the sample solution at ahigh concentration, and other trace components, C1, C2… and Cn, diameters in the longand short axial directions of the inhibition zones of all these components are measured
to the 0.1 mm with a caliper. The mean of the values is recognized as a diameter of theinhibition zone. To prepare the standard curve, the potencies of monensin in thestandard solution S2, S3, and S4, are plotted on the logarithmic axis and the diametersof the inhibition zones of monensin A in the standard solutions are plotted on the536

Provisional Translation from Japanese Original

integral axis. The potency of monensin A is measured using the standard curve as formonensin A, B, C1, C2… and Cn, in the sample solutions.
Calculation: The potency ratio of monensin A is calculated by the following formula.
Potency ratio of monensin A (%) =

10PA
× 100
10PA + PB + PC1 … … + PCn

PA: Potency of monensin A in the sample solution at a low concentration

PB: Potency of monensin B in the sample solution at a high concentration as monensinA
PC1 to Cn: Potency of trace components C1~Cn in the sample solution at a highconcentration as monensin A
ⅲ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 3 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ⅳ. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
Loss on drying: 4.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 8.0~13.0 % (1 g)
Potency test:
Agar plate (single layer): 10 mL of the No. 14 medium mixed with the test strain is used
(11 mL in a Petri dish with internal diameter of 100 mm).
Test strain: Bacillus subtilis ATCC 6633 is used.
Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is dissolved with methanol, and is further added with methanol to make aconstant volume at a concentration of approximately 1 mg/mL (potency). This is used
as a diluted stock solution. The volume required for testing of the diluted stock
solution is measured using a volumetric pipette, accurately diluted with water andmethanol to prepare the working standard diluent at a high concentration of 40 µg/mL(potency) with 10 % methanol and the working standard diluent at a low concentrationof 10 µg/mL (potency) with 10 % methanol.
Preparation of sample solution: The amount of the working reference standard required
for testing is weighed to three significant digits and the value is recorded. It is537

Provisional Translation from Japanese Original

dissolved with methanol, and is further added with methanol to make a constantvolume at a concentration (estimated value) of approximately 1 mg/mL (potency).
This is used as a sample stock solution. The volume required for testing of the sample
stock solution is measured using a volumetric pipette, accurately diluted with waterand methanol to prepare the sample solution at a high concentration of 40 µg/mL(potency) with 10 % methanol and the sample solution at a low concentration of 10µg/mL (potency) with 10 % methanol.
(b) Standard of manufacturing method
For manufacturing: The monensin producing strain of Streptomyces cinnamonensis isaerobically cultured, after the cultivation the pH of the culture solution is adjusted andmonensin is extracted with an organic solvent. The solution is concentrated and filteredand crystallized as sodium salt under alkaline conditions. The obtained crystals are
washed and dried.
(c) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is small pieces, powder or particles of mixture of the raw material for
manufacturing of monensin sodium and carriers.
Potency: When this product is conducted potency test, it contains 240 µg (potency) or lessin 1 mg including 85~125 % of the indicated potency.
Physical and chemical properties:
ⅰ. This product is light grayish brown to brown or yellowish white to yellow small pieces,
powder or particles with a slightly specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:ⅰ. According to the label potency of this product, the amount containing approximately 10
mg (potency) of monensin is weighed, added with 10 mL of methanol, shaken andfiltered. This solution 5 mL is measured, and the confirmation test i. of the raw
material for manufacturing of monensin sodium is hereinafter applied mutatis
mutandis.
ⅱ. According to the label potency of this product, the amount containing approximately
10 mg (potency) of monensin is weighed, added with 10 mL of ethanol, shaken andfiltered. This solution 5 mL is measured, and the confirmation test ii. of the raw

538

Provisional Translation from Japanese Original

material for manufacturing of monensin sodium is hereinafter applied mutatis
mutandis.
Loss on drying: 12.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate: The provisions of the raw materials for manufacturing of monensin sodium
are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of monensin sodium
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of monensin sodium are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount required for testing is weighed to three significant digits and the value isrecorded. It is added with methanol using a volumetric pipette to make its
concentration of 1~2 mg/mL (potency), stirred or shaken, and then filtered or
centrifuged. The filtrate or supernatant is used as a sample stock solution. The volume
required for testing of the sample stock solution is measured using a volumetricpipette, and the provisions of the raw material for manufacturing of monensin sodium
are hereinafter applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the raw material for manufacturing of monensin sodium is mixed
with carriers, and as appropriate granulated.
(c) Standard of storage method
The provisions of the raw materials for manufacturing of monensin sodium are appliedmutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture“使用上の注意 この飼料添加物は、鶏又は牛に過剰投与した場合発育障害がおこるので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”Precautions: Overdose of this feed additive causes developmental disorders in chicken or
cattle, so strictly follow the specified amount to be added and make sure to mix it
uniformly.

539

Provisional Translation from Japanese Original(124) Lasalocid Sodium
A. Raw material for manufacturing(a) Compositional standards
Potency: This product is sodium salt of lasalocid. The potency test shows that 800 µg(potency) or more is contained in 1 mg.
Physical and chemical properties:
ⅰ. This product is white to band brown white powder with a specific odor.
ⅱ. This product is hard to dissolve in acetone, ethanol, choloroform, ethyl acetate or
methanol and hardly dissolves in water.
Confirmation test:
ⅰ. 10 mL of absolute ethanol solution of this product (1 → 500) is shaken with 0.5 mL ofabsolute ethanol solution of ferric chloride (1 → 50). The resulting solution is reddish
violet.
ⅱ. 75 mg (74.5~75.4 mg) of this product is weighed and dissolved with a mixed solutionof sulfuric acid and absolute ethanol (3: 1,000) to make 50 mL. 5 mL of this solution ismeasured and added with a mixed solution of sulfuric acid and absolute ethanol (3:1,000) to make 100 mL. In the measurement of the absorption spectrum of thesolution, the absorption maximum is at the wavelengths of 245~249 nm and 315~319
nm, the absorption minimum is at the wavelengths of 269~273 nm.
ⅲ. The ethyl acetate solution of this product (1 → 200) irradiated with ultraviolet light(dominant wavelength: 365 nm) exhibits blue fluorescence.
ⅳ. 3 g (2.5~3.4 g) of this product is ashed, shaken with 10 mL of water, and filtered. The
resulting solution gives the qualitative reaction of sodium salt.
Purity test:
ⅰ. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed to prepare a sample
solution by method No. 2 of the heavy metals test method. When heavy metal is tested
using the sample solution and a control solution prepared using 2.0 mL of leadstandard solution, the color of the sample solution shall not be darker than that of thecontrol solution (20 mg/kg or less).
ⅱ. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed and the sample solution isprepared by method No. 3 of the arsenic test method. The arsenic test is performed by
the method using device A. The color of the absorbing solution shall not be darker
than the standard color (4 mg/kg or less).
Loss on drying: 3.0 % or less (1 g, 0.67 kPa or less, 60 °C, 3 hours)
Ignition residue: 10.0~15.0 % (1 g)
Potency test:540

Provisional Translation from Japanese Original

Agar plate: Medium No. 14 is used for both the media of base and inoculated layers.
Test strain: Bacillus subtilis ATCC 6633 is used.
Preparation of the working standard diluent: The amount of the working reference
standard required for testing is weighed to three significant digits and the value isrecorded. It is dissolved with methanol, and is further added with methanol to make aconstant volume at a concentration of 1 mg/mL (potency). This is used as a diluted
stock solution. The volume required for testing of the diluted stock solution ismeasured using a volumetric pipette and accurately diluted by adding water andmethanol to prepare the working standard diluent at a high concentration of 40 µg/mL(potency) with 10 % methanol and the working standard diluent at a low concentrationof 10 µg/mL (potency) with 10 % methanol.
Preparation of the sample solution: The amount of this product required for testing isweighed to three significant digits and the value is recorded. It is dissolved withmethanol to make an accurate constant volume with the concentration (estimatedvalue) of approximately 1 mg/mL (potency). This is used as a sample stock solution.
The volume required for testing of the sample stock solution is measured using a
volumetric pipette and accurately diluted by adding water and methanol to prepare thesample solution at a high concentration (estimated value) of 40 µg/mL (potency) with
10 % methanol and the sample solution at a low concentration (estimated value) of 10µg/mL (potency) with 10 % methanol.
(b) Standard of manufacturing method
The lasalocid producing strain of Streptomyces lasaliensis is aerobically cultured, after
the cultivation, the culture solution is added with a small amount of a filter aid, and thesolids are separated by filtration. Lasalocid in the solids is extracted using an organic
solvent, and the extract is concentrated, dissolved with a different organic solvent, washed,
and filtered and crystallized as a sodium salt under alkaline conditions. The resulting
crystals are washed and dried.
(c) Standard of storage method
It shall be stored in a sealed container.
B. Preparation(a) Compositional standards
This product is powder or particles in which the raw material for manufacturing lasalocidsodium is mixed with carriers.
Potency: The potency test shows that 150 µg (potency) or less is contained in 1 mg, and thatit contains 85~125 % of the label potency.
Physical and chemical properties:

541

Provisional Translation from Japanese Originalⅰ. This product is pale brown to brown small powder or particles with a specific odor.
ⅱ. This product passes through a standard 2.00 mm mesh sieve.
ⅲ. There is no mold in this product.
Confirmation test:ⅰ. According to the label potency of this product, the amount containing approximately
100 mg (potency) of lasalocid sodium is weighed, added with 50 mL of absoluteethanol, shaken well for 10 minutes, and filtered. The filtrate 10 mL is shaken with adrop of absolute ethanol solution of ferric chloride (1 → 50), and the resulting solution
is reddish violet.
ⅱ. According to the label potency of this product, the amount containing approximately
20 mg (potency) of lasalocid sodium is weighed, added with 10 mL of methanol,
shaken well for 10 minutes, and filtered. The filtrate is used as a sample solution.
Separately, the amount of working standard lasalocid containing approximately 20 mg(potency) of lasalocid sodium is weighed, dissolved with 10 mL of methanol. This isused as a standard solution. 10 µL each of the sample and standard solutions are
spotted on a thin layer plate prepared using silica gel with a fluorescent agent for thinlayer chromatography. Next, they are developed to approximately 10 cm using amixed solution of chloroform and methanol (9:1) as the developing solvent and thethin layer plate is air-dried. When it is irradiated with ultraviolet light (dominant
wavelength: 254 nm), the spots of the sample and standard solutions are dark-blue andthe Rf values are equal.
Loss on drying: 15.0 % or less (1 g, 105 °C, 3 hours)
Potency test:
Agar plate: The provisions of the raw materials for manufacturing of lasalocid sodium
are applied mutatis mutandis.
Test strain: The provisions of the raw materials for manufacturing of lasalocid sodium
are applied mutatis mutandis.
Preparation of the working standard diluent: The provisions of the raw materials for
manufacturing of lasalocid sodium are applied mutatis mutandis.
Preparation of sample solution: According to the label potency of this product, theamount required for testing is weighed to three significant digits and the value isrecorded. It is added with a constant volume of methanol (9 → 10) using a volumetricpipette to make its concentration approximately 1 mg/mL (potency), vigorously
shaken, and if necessary filtered or centrifuged. The resulting filtrate or supernatant isused as a sample stock solution. The volume required for testing of the sample stock

542

Provisional Translation from Japanese Original

solution is measured by a volumetric pipette, and the provisions of the raw material for
manufacturing of lasalocid sodium are hereinafter applied mutatis mutandis.
(b) Standard of manufacturing method
The raw material for manufacturing of lasalocid sodium is mixed with carriers.
(c) Standard of storage method
The storage method of the raw materials for manufacturing of lasalocid sodium areapplied mutatis mutandis.
(d) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“有効期間 製造の翌月から２年”
Valid period: 2 years from the month following the manufacture“使用上の注意 この飼料添加物は、鶏又は牛に過剰投与した場合発育障害がおこるので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”Precautions: Overdose of this feed additive causes developmental disorders in chicken or
cattle, so strictly follow the specified amount to be added and make sure to mix it
uniformly.

(125) Amprolium plus Ethopabate
A. Raw material for manufacturing(a) Compositional standards
Amprolium
Content: When this product is determined, it contains 95.0 % or over of amprolium(C14H19ClN4·HCl).
Physical and chemical properties:
i. This product is white to pale yellow powder with no odor or a slight specific odor.
ii. This product is easy to dissolve in water, easy to slightly dissolve in methanol, hard todissolve in ethanol, and hardly dissolves in ether or chloroform.
Confirmation test:
i. In the measurement of the absorption spectrum of 0.1 mol/L hydrochloric acid solutionof this product (1 → 100,000), the absorption maximum is at the wavelengths of
244~248 nm and 260~264 nm. The absorbances at both wavelengths are referred to as
A1 and A2 respectively, and the A1 / A2 is 1.04~1.06.

543

Provisional Translation from Japanese Original

ii. The solution of this product in water (1 → 50) gives the qualitative reaction ofchloride.
Purity test:
i. Picoline:
Device: To the side-arm of a 100 mL side-arm distilling flask, a 200 mL receiver isconnected so that the tip almost contacts with the bottom. The mouth of the
distilling flask is connected to a dropping funnel and an air tube so that the lower
end is near the bottom of the flask. The other end of the air tube is sequentiallyconnected to two scrubbing bottles, and glass fibers and sulfuric acid are placed sothat the one directly connects to the air tube and the other one, respectively.
Procedure: Approximately 1.0 g of this product is weighed to the digits of 0.01 g andthe value is recorded. It is placed in a side-arm distilling flask and dissolved with 10
mL of water. Boric acid solution (1 → 50) 75 mL and 0.5 mL of bromcresol green,
methyl orange test solution are transferred to a receiver and the tip of the connectedside-arm is dipped in the solution. A dropping funnel and air tube are attached. Withadding 25 mL of saturated solution of potassium carbonate by dropping from thedropping funnel for approximately 10 minutes and aeration through the scrubbing
bottles for approximately 1 hour, picoline present in the solution is collected in thesolution in the receiver. Then, the aeration is stopped, the receiver is replaced withanother receiver with 75 mL of boric acid solution (1 → 50) and 0.5 mL ofbromcresol green, methyl orange test solution, and again aeration is performed for
30 minutes. The color of solution in the receiver is changed, the receiver is also
replaced. This procedure is repeated, and stopped when there is no change in colorof the solution in the receiver. When total solution in all receivers is titrated with
0.1 mol/L hydrochloric acid to determine the amount of picoline (C6H7N), theamount shall be 1.0 % or less. In this case, the end of titration is the time whengreen-blue changes to yellow-green to yellow.
1 mL of 0.1 mol/L hydrochloric acid = 9.313 mg C6H7N
ii. Chlorine: Approximately 0.15 g of this product is weighed to the digits of 0.001 g andthe value is recorded. It is dissolved with 50 mL of water, added with 5 mL of nitricacid, added with 25 mL of 0.1 mol/L of silver nitrate solution using a volumetricpipette, mixed, added with 3 mL of nitrobenzene, vigorously shaken, and added with 2
mL of ferric ammonium sulfate test solution. The excess silver nitrate is titrated with
0.1 mol/L of ammonium thiocyanate to determine the amount of chlorine (Cl), and theamount shall be 21.5~23.5 %.
1 mL of 0.1 mol/L silver nitrate solution = 3.545 mg Cl

544

Provisional Translation from Japanese Original

Loss on drying: 1.0 % or less (1 g, reduced pressure, 100 °C, 3 hours)
Ignition residue: 0.20 % or less (1 g)Assay: 0.05 g of this product is weighed to the digits of 0.1 mg and the value is recorded. It
is dissolved in methanol (2 → 3), transferred to a 100 mL volumetric flask, is further
added with methanol (2 → 3) to the graduation linegraduation line to make 100 mL. Thissolution 5 mL is measured with a volumetric pipette, transferred to a 100 mL volumetricflask, added with methanol (2 → 3) to the graduation linegraduation line to make 100
mL. This is used as a sample solution. Separately, appropriately 0.05 g of driedamprolium reference standard is weighed to the digits of 0.1 mg and the value isrecorded. It is dissolved in methanol (2 → 3), transferred to a 100 mL volumetric flask, isfurther added with methanol (2 → 3) to the graduation linegraduation line to make 100
mL. 5 mL of this solution is measured with a volumetric pipette, transferred to a 100 mL
volumetric flask, added with methanol (2 → 3) to the graduation line to make 100 mL.
This is used as a standard solution. 4 mL each of the sample and standard solutions aremeasured using a volumetric pipette, transferred to test tubes (1) and (2), and separately,
4 mL of methanol (2 → 3) is measured using a volumetric pipette and transferred to a test
tube (3). Each of the test tubes is added with 10 mL of 2,7-dioxynaphthalene using a
volumetric pipette, tightly stoppered, mixed well, and allowed to stand for 20 minutes.
Then, each tube is centrifuged for 2~3 minutes, and the supernatants of (1) and (2) areadded with 2,7-dioxynaphthalene test solution, in 20~45 minutes, the absorbances AT
and AS at the maximum wavelength near 530 nm are measured using the supernatant of(3) as a control.
Amount of amprolium (C14H19 CIN4ꞏHCl) (mg)
= Amount of amprolium standard (mg)AT
AS

Ethopabate
Content: When this product is determined, it contains 95.0~103.0 % of ethopabate(C12H15NO4).
Physical and chemical properties:
i. This product is white to pale red-white powder and with a slight or no odor.
ii. This product is easy to dissolve in chloroform, easy to slightly dissolve in methanol or
ethanol, hard to extreamly dissolve in ether, and hardly dissolves in water.
Confirmation test:
i. In the measurement of the absorption spectrum of the methanol solution (1 → 125,000)of this product, the absorption maximum is at the wavelengths of 266~270 nm and545

Provisional Translation from Japanese Original

297~301 nm, and the absorption minimum is at the wavelengths of 236~240 nm and285~289 nm.
ii. This product and ethopabate reference standard are dried, measured by the potassium
bromide disk method of the infrared absorption spectroscopy. When their spectra arecompared, both absorptions are observed at the same wavenumber and the relative
intensities of these absorptions are equal.
Purity test:
i. Melting point: The melting point of this product shall be 146~151 °C.
ii. Diazotized substance: Approximately 1.0 g of this product is weighed to the digits of
0.01 g and the value is recorded. It is dissolved with chloroform, transferred to a 100mL volumetric flask, and is further added with chloroform to the graduation line to
make 100 mL. This solution 10 mL is measured using a volumetric pipette, added with
100 mL of 0.1 mol/L hydrochloric acid using a volumetric pipette and shaken, and theaqueous layer is sufficiently separated to prepare the first extract. The chloroform layer
is added with 100 mL of 0.1 mol/L hydrochloric acid using a volumetric pipette andshaken, and the aqueous layer is separated to prepare the second extract. 40 mL eachof the first and second extracts are centrifuged and 20 mL each of the supernatants ismeasured using a volumetric pipette are mixed to prepare a sample solution.
Separately, 4-amino-2-ethoxybenzoic acid methyl reference standard is dried under
reduced-pressure at 100 °C for 2 hours and approximately 0.05 g of it is weighed to thedigits of 0.1 mg and the value is recorded. It is dissolved with chloroform, transferred
to a 100 mL volumetric flask, and is further added with chloroform to the graduationline to make 100 mL. 2 mL of this solution is measured using a volumetric pipette,added with 8 mL of chloroform, added with 100 mL of 0.1 mol/L hydrochloric acidusing a volumetric pipette and shaken, and the same procedure as that for the sample
solution is hereinafter taken to prepare a standard solution. Five stoppered test tubes(1) to (5) are prepared. 5 mL of the sample solution, standard solution 1 mL and 0.1
mol/L hydrochloric acid 4 mL, standard solution 3 mL and 0.1 mol/L hydrochloric
acid 2 mL, standard solution 5 mL, 0.1 mol/L hydrochloric acid 5 mL are measured
using a volumetric pipette and transferred to the stoppered test tubes (1), (2), (3), (4),and (5), respectively. Each stoppered test tube is subjected to the following procedure.
Into each tube, 6 mL of 1 mol/L hydrochloric acid and 1 mL of newly prepared sodium
nitrite solution (1 → 1,000) are added using a volumetric pipette, shaken, and allowed
to stand for 4 minutes. Then it is added with 1 mL of newly prepared ammonium
sulfamate solution (1 → 200) measured using a volumetric pipette, shaken, andallowed to stand for 3 minutes. Next, it is added with 1 mL of newly prepared N-(1-546

Provisional Translation from Japanese Original

naphthyl)-ethylenediamine dihydrochloride solution (1 → 1,000) using a volumetricpipette, shaken, and allowed to stand for 30 minutes. The absorbances A1, A2, A3 andA4 of the solutions of the tubes (1) to (4) are measured at the maximum wavelength
near 530 nm, using the solution of the tube (5) as a control. The calibration curves areprepared from the absorbances A2, A3 and A4 and the amounts (mg) of the reference
standard in the corresponding 1 mL, 3 mL and 5 mL of standard solutions. The amount(mg) of the diazotized substance [as 4-amino-2-ethoxybenzoic acid methyl(C10H13NO3)] in the sample solution 5 mL corresponding to the absorbance A1 shall be
1.0 % or less.
iii. Phenolic substance: Approximately 0.4 g of this product is weighed to the digits of
0.001 g and the value is recorded. It is dissolved with methanol, transferred to a 20 mL
volumetric flask, and is further added with methanol to the graduation line to make 20
mL. This is used as a sample solution. Separately, 4-acetamide-2-hydroxybenzoic acidmethyl reference standard is dried and approximately 0.028 g of it is weighed to thedigits of 0.1 mg and the value is recorded. It is dissolved with methanol, transferred toa 100 mL volumetric flask, and is further added with methanol to the graduation line to
make 100 mL. This is used as a standard solution. Nine centrifugal precipitation tubes(1) to (9) are prepared. 1 mL of the sample solution and 4 mL of methanol, 3 mL ofsample solution and 2 mL of methanol, 5 mL of sample solution, 1 mL of standard
solution and 4 mL of methanol, 2 mL of standard solution and 3 mL of methanol, 3
mL of standard solution and 2 mL of methanol, 4 mL of standard solution and 1 mL of
methanol, 5 mL of standard solution, and 5 mL of methanol are measured using a
volumetric pipette and placed in the centrifugal precipitation tubes (1) to (9),respectively. Each centrifugal precipitation tube is added with 5 mL of ferricperchloric acid test solution using a volumetric pipette, shaken, allowed to stand for 10
minutes. The centrifugal precipitation tubes (1), (2), and (3) are centrifuge to separate
the supernatant. The absorbances A1 to A8 of the solutions of (1) to (8) are measured at
the maximum wavelength near 525 nm, using the solution of (9) as a control. The
calibration curves are prepared from the absorbances A4 to A8 and the amounts (mg) of
the reference standard in the corresponding 1 to 5 mL of standard solutions. The
amount (mg) of the phenolic substance [as 4-acetamide-2-hydroxybenzoic acid methyl(C10H11NO4)] in the sample solution 1, 3 and 5 mL corresponding to the absorbance A1A2 and A3 is determined. The average of the amounts of phenolic substancedetermined by respective sample solutions shall be 2.0 % or less.
Loss on drying: 1.0 % or less (1 g, reduced pressure, 100 °C, 2 hours)
Ignition residue: 0.5 % or less (1 g)

547

Provisional Translation from Japanese Original

Assay: Approximately 0.075 g of this product is weighed to the digits of 0.1 mg and thevalue is recorded. It is dissolved in methanol, transferred to a 250 mL volumetric flask,
and is further added with methanol to the graduation line to make 250 mL. 10 mL of thissolution is measured using a volumetric pipette, transferred to a 100 mL volumetric flask
and added with water to the graduation line to make 100 mL. 10 mL of this solution istransferred to a beaker using a volumetric pipette, added with 10 mL of 1 mol/L sodiumhydroxide test solution, and evaporated to dryness in a water bath. It is added with 10 mLof boiling water while washing the inside wall, and is further heated for 15 minutes. Aftercooling, it is added with water to make 20 mL and transferred to a 100 mL volumetricflask. The beaker is washed three times with 20 mL of water each. The washings are putin the volumetric flask and more water is added to the graduation line to make 100 mL.
This is used as a sample solution. Separately, ethopabate reference standard is dried,
approximately 0.03 g of it is weighed to the digits of 0.1 mg and the value is recorded. It
is dissolved in methanol, transferred to a 100 mL volumetric flask, and is further addedwith methanol to the graduation line to make 100 mL. 10 mL of this solution is measured
using a volumetric pipette, transferred to a 100 mL volumetric flask and added with water
to the graduation line to make 100 mL. 10 mL of this solution is transferred to a beakerusing a volumetric pipette, added with 10 mL of 1 mol/L sodium hydroxide test solution,and the same procedure as that for the sample solution is hereinafter performed to preparea standard solution. Separately, 10 mL of water is measured using a volumetric pipetteand transferred to a beaker, added with 10 mL of 1 mol/L sodium hydroxide test solution,and the same procedure as that for the sample solution is hereinafter performed to preparea blank test solution. Three centrifugal precipitation tubes (1) to (3) are prepared. 10 mLof the sample solution, 10 mL of the standard solution and 10 mL of the blank test
solution are measured using a volumetric pipette and transferred to the centrifugal
precipitation tubes (1), (2) and (3), respectively. Each precipitation tube is subjected to
the following procedure. Each precipitation tube is added with 2 mL of 1 mol/Lhydrochloric acid and 2 mL of newly prepared sodium nitrite solution (1 → 1,000) using
a volumetric pipette, shaken and allowed to stand for 2 minutes. Then, it is added with 2
mL of newly prepared sulfamic acid ammonium solution (1 → 200) measured using a
volumetric pipette, shaken and allowed to stand for 2 minutes. It is added with 2 mL of
newly prepared N-(1-naphthyl)-ethylenediamine dihydrochloride (1 → 1,000) measured
using a volumetric pipette, shaken and allowed to stand for 10 minutes. It is added with 4
g (3.5~4.4 g) of sodium chloride, and added with 10 mL of n-butanol measured using a
volumetric pipette. It is vigorously shaken for 2 minutes and centrifuged to separate the548Provisional Translation from Japanese Original

supernatant. The absorbances AT and AS of the solution (1) and (2) at the maximum
wavelength near 555 nm are measured, using the solution (3) as a control solution.
Amount of ethopabate (C12H15 NO4) (mg)
= Amount of ethopabate reference standard (mg)

AT
× 2.5
AS

- Amount of corresponding diazotized substance (mg) × 1.215- Amount of corresponding phenolic substance (mg) × 1.134(b) Standard of storage method
Amprolium
It shall be stored in a capped container.
Ethopabate
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
This product is solid or powder, in which the raw material for manufacturing of formic
acid or its preparation and the raw material for manufacturing of propionic acid or its
preparation are mixed and if necessary added with ammonia (limited to that meeting thestandards of food additives), caprylic acid or food dye.
Content: This product is determined to contain amprolium (C14H19ClN4·HCl) andethopabate (C12H15NO4) corresponding to 90~110 % of the amount on the label.
Confirmation test:
i. The sample solution obtained by the assay of amprolium is reddish violet and theabsorption maximum is at the wavelengths of 528~532 nm by measuring theabsorption spectrum.
ii. The sample solution obtained by the assay of ethopabate is reddish violet and theabsorption maximum is at the wavelengths of 553~557 nm by measuring theabsorption spectrum.
Assay:
Amprolium: The amount of this product containing approximately 0.05 g of amprolium(C14H19ClN4·HCl) is weighed to three significant digits and the value is recorded. It is
added with 100 mL of methanol (2 → 3) using a volumetric pipette and shaken for 20
minutes. This solution is filtered, the first filtrate 20 mL is removed, and the next
filtrate 5 mL is measured using a volumetric pipette, transferred to a 100 mL
volumetric flask, added with methanol (2 → 3) to the graduation line to make 100 mL,
and the assay of the raw material for manufacturing of amprolium is hereinafter
applied mutatis mutandis.

549

Provisional Translation from Japanese OriginalAmount of amprolium (C14H19 ClN4ꞏHCl) (mg)
= Amount of amprolium standard (mg) ×

AT
AS

Ethopabate: The amount of this product containing approximately 3 mg of ethopabate(C12H15NO4) is weighed to three significant digits and the value is recorded. It is placedin a 100 mL volumetric flask, added with 75 mL of methanol, shaken for 20 minutes, isfurther added with methanol to the graduation line to make 100 mL, and centrifuged. The
supernatant 10 mL is transferred to a beaker using a volumetric pipette, added with 10
mL of sodium hydroxide test solution, and the assay of the raw material for
manufacturing of ethopabate is hereinafter applied mutatis mutandis.
Amount of ethopabsate (C12H15NO4) (mg)
= Amount of ethopabsate standard (mg ) ×

AT
1
×
AS 10

(b) Standard of storage method
It shall be stored in a capped container.

(126) Amprolium/ethopabate/sulfaquinoxaline
A. Raw material for manufacturing(a) Compositional standards
Amprolium
The compositional standards of the raw material for manufacturing of Amprolium in
Amprolium plus Ethopabate are applied mutatis mutandis.
Ethopabate
The compositional standards of the raw material for manufacturing of Ethopabate in
Amprolium plus Ethopabate are applied mutatis mutandis.
Sulfaquinoxaline
Content: When this product is determined after dried, it contains 98.0 % or over ofsulfaquinoxaline (C14H12N4O2S).
Physical and chemical properties:
i. This product is pale yellow to yellowish-brown crystalline powder with little odor.
ii. This product is hard to dissolve in acetone, extremely hard to dissolve in ethanol andhardly dissolves in water.
iii. This product is dissolved in sodium carbonate test solution or 1 mol/L sodiumhydroxide test solution.
iv. This product gradually becomes dark in color by light.

550

Provisional Translation from Japanese OriginalConfirmation test:
i. 0.05 g (0.045~0.054 g) of this product is dissolved with 4 mL of dilute hydrochloric byheating. The resulting solution gives the qualitative reaction of aromatic primaryamine, however, its color is orange-red.
ii. 0.02 g (0.015~0.024 g) of this product is weighed, added with 5 mL of water, addedwith 1 mol/L sodium hydroxide by dropping while stirring to dissolve, and added with
2~3 drops of copper sulfate test solution, and then yellow-green precipitation develops.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved with 5 mLof 1 mol/L sodium hydroxide test solution, and is further added with 20 mL of water.
The resulting solution shall be orange-yellow and clear.
ii. Melting point: The melting point of this product shall be 244~247 °C (Disassembly)iii. Acid: 1.0 g (0.95~1.04 g) of this product is weighed, added with 50 mL of water,heated at 70 °C for 5 minutes, quenched to room temperature, and filtered. The filtrate
25 mL is added with 2 drops of methyl red test solution and 0.50 mL of 0.1 mol/L
sodium hydroxide solution. The resulting solution shall be yellow.
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (20 mg/kg or
less).
Loss on drying: 1.0 % or less (1 g, 105 °C, 4hours)
Ignition residue: After drying, 0.10% or less (1 g)Assay: This product is dried and approximately 0.5 g of it is weighed to the digits of 0.001 g
and the value is recorded. It is dissolved with 75 mL of glacial acetic acid, 8 mL ofhydrochloric acid, and 25 mL of water. It is cooled to 15 °C, added with 25 g (24.5~25.4g) of crushed ice, titrated with 0.1 mol/L sodium nitrite solution with constant stirring. In
this case, the end of titration is the time when the titrated solution is put on a glass rod 1
minute after dropping 0.1 mol/L sodium nitrite solution, the tip of the glass rod attaches
to zinc iodide-starch test paper, and it turns blue within 30 seconds. In the same manner, ablank test is performed and corrections are made.
(b) Standard of storage method
Amprolium: The standard of storage method of the raw material for manufacturing of
Amprolium in Amprolium plus Ethopabate are applied mutatis mutandis.

551

Provisional Translation from Japanese Original

Ethopabate: The standard of storage method of the raw material for manufacturing ofEthopabate in Amprolium plus Ethopabate are applied mutatis mutandis.
Sulfaquinoxaline: It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Amprolium,ethopabate and sulfaquinoxaline and fillers.
Content: This product is determined to contain amprolium (C14H19ClN4·HCl) , ethopabate(C12H15NO4) and sulfaquinoxaline (C14H12N4O2S) corresponding to 90~110 % of theamount on the label.
Confirmation test:
i. The sample solution obtained by the assay of amprolium is reddish violet and theabsorption maximum is at the wavelengths of 528~532 nm by measuring theabsorption spectrum.
ii. The sample solution obtained by the assay of ethopabate is reddish violet and theabsorption maximum is at the wavelengths of 538~542 nm by measuring theabsorption spectrum.
iii. The sample solution obtained by the assay of sulfaquinoxaline is reddish violet andthe absorption maximum is at the wavelengths of 543~547 nm by measuring theabsorption spectrum.
Assay: Amprolium: The amount of this product containing approximately 0.05 g ofamprolium (C14H19ClN4·HCl) is weighed to three significant digits and the value isrecorded. It is added with 100 mL of methanol (2 → 3) using a volumetric pipette andshaken for 20 minutes. This solution is filtered, the first filtrate 20 mL is removed, andthe second filtrate 5 mL is measured using a volumetric pipette, transferred to a 100 mL
volumetric flask, added with methanol (2 → 3) to the graduation line to make 100 mL,
and the assay of the raw material for manufacturing of amprolium is hereinafter appliedmutatis mutandis.
Amount of amprolium (C14H19 CIN4ꞏHCl) (mg)
= Amount of amprolium standard (mg)AT
AS

Ethopabate: The amount of this product containing approximately 6 mg of ethopabate(C12H15NO4) is weighed to three significant digits and the value is recorded. It is placedin a saponification flask, added with 100 mL of chloroform using a volumetric pipette.
The flask is attached with a reflux condenser and the reflux is performed in a water bathfor 15 minutes, avoiding chloroform evaporation. After cooling, it is filtered. The filtrate552

Provisional Translation from Japanese Original

50 mL is measured using a volumetric pipette, placed in a separatory funnel, washedthree times with 25 mL each of sodium carbonate solution (1 → 20), and then washedtwice with 10 mL of water, and the washings are discarded. The chloroform layer isfiltered and 10 mL of the filtrate is measured using a volumetric pipette, almost
evaporated in a water bath, added with 10 mL of methanol and 10 mL of 1 mol/L sodiumhydroxide test solution and evaporated to dryness in a water bath. It is added with 10 mLof boiling water, heated for 15 minutes, allowed to cool, transferred to a 100 mL
volumetric flask, added with 20 mL of 1 mol/L hydrochloric acid and water to thegraduation line to make 100 mL, and filtered. This is used as a sample solution.
Separately, ethopabate reference standard is dried, and approximately 0.03 g of it isweighed to the digits of 0.1 mg and the value is recorded. It is dissolved with methanol,transferred to a 100 mL volumetric flask, and is further added with methanol to thegraduation line to make 100 mL. 10 mL of this solution is measured by a volumetricpipette, transferred to a 50 mL volumetric flask, and added with methanol to thegraduation line to make 50 mL. 10 mL of this solution is measured using a volumetricpipette, added with 10 mL of 1 mol/L sodium hydroxide test solution, and the sameprocedure as that for the sample solution is hereinafter performed. This is used as astandard solution. Separately, 10 mL of methanol is measured, added with 10 mL of 1
mol/L sodium hydroxide test solution, and hereinafter subjected to the same procedure asthat for the sample solution to prepare a blank test solution.
Three 50 mL volumetric flasks (1) to (3) are prepared, and 25 mL each of the sample
solution, the standard solution, and the blank test solution are measured using a
volumetric pipette and transferred to the 50 mL volumetric flasks (1), (2), and (3),respectively. Then each of them is subjected to the following procedure. Each solution isadded with 5 mL of 1 mol/L hydrochloric acid and 5 mL of newly prepared sodium
nitrite solution (1 → 1,000) using a volumetric pipette, shaken, and allowed to stand for 2
minutes. It is added with 5 mL of newly prepared sulfamic acid ammonium solution (1→ 200) using a volumetric pipette, shaken, allowed to stand for 3 minutes, added with 5
mL of newly prepared N-(1-naphthyl)-ethylenediamine dihydrochloride solution (1 →1,000) using a volumetric pipette, shaken, allowed to stand for 10 minutes, and thenadded with water to the graduation line to make 50 mL. The absorbances AT and AS of
the solution (1) and (2) at the maximum wavelength near 540 nm are measured, using thesolution (3) as a control solution.
Amount of ethopabate (C12H15 NO4 ) (mg)
= Amount of amprolium standard (mg)

AT 1
×
AS 5

553

Provisional Translation from Japanese Original

Sulfaquinoxaline: The amount of this product containing approximately 0.05 g ofsulfaquinoxaline (C14H12N4O2S) is weighed to three significant digits and the value isrecorded. It is added with 70 mL of water and 10 mL of 0.5 mol/L sodium hydroxide test
solution, shaken for 10 minutes, transferred to a 250 mL volumetric flask, and added withwater to the graduation line to make 250 mL. It is allowed to stand for 2 minutes, and if
necessary centrifuged. 10 mL of the supernatant is measured using a volumetric pipette,
transferred to a 200 mL volumetric flask with 10 mL of 1 mol/L hydrochloric acid, andadded with water to the graduation line to make 200 mL. This is used as a sample
solution. Separately, sulfaquinoxaline reference standard is dried, and approximately 0.05
g of it is weighed to the digits of 0.1 mg and the value is recorded. It is dissolved with 70
mL of water and 10 mL of 0.5 mol/L sodium hydroxide test solution, transferred to a 250
mL volumetric flask, and added with water to the graduation line to make 250 mL. Thissolution 10 mL is measured using a volumetric pipette, transferred to a 200 mL
volumetric flask with 10 mL of 1 mol/L hydrochloric acid, and added water to thegraduation line to make 200 mL. This is used as a standard solution. Separately, 10 mL of
0.5 mol/L sodium hydroxide test solution is added with water to make 250 mL and 10 mLof this solution is transferred to a 200 mL volumetric flask and added with 10 mL of 1
mol/L hydrochloric acid and water to the graduation line to make 200 mL. This is used asa blank test solution. Three 50 mL volumetric flasks (1) to (3) are prepared, and 10 mL
each of the sample solution, the standard solution, and the blank test solution aremeasured using a volumetric pipette and transferred to the 50 mL volumetric flasks (1),(2), and (3), respectively. Then each of them is subjected to the following procedure.
Each solution is added with 5 mL of 1 mol/L hydrochloric acid and 5 mL of newly
prepared sodium nitrite solution (1 → 1,000) using a volumetric pipette, shaken, andallowed to stand for 2 minutes. It is added with 5 mL of newly prepared sulfamic acidammonium solution (1 → 200) using a volumetric pipette, shaken, allowed to stand for 3
minutes, added with 5 mL of newly prepared N-(1-naphthyl)-ethylenediamine
dihydrochloride solution (1 → 1,000) using a volumetric pipette, shaken, allowed tostand for 10 minutes, and then added with water to the graduation line to make 50 mL.
The absorbances AT and AS of the solution (1) and (2) at the maximum wavelength near
545 nm are measured, using the solution (3) as a control solution.
Amount of sulfaquinoxaline (C14 H12 N4O2S) (mg)
= Amount of amprolium standard (mg) ×(b) Standard of storage method
It shall be stored in a capped container.

554

A
AS

Provisional Translation from Japanese Original(127) Morantel citrate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 98.0 % or over of Morantel citrate(C12H16N2S·C6H8O7·H2O).
Physical and chemical properties:
i. This product is pale-yellow to yellow crystalline powder, with a slightly bitter taste anda specific odor.
ii. This product is easy to slightly dissolve in methanol, hard to dissolve in ethanol andwater, and hardly dissolves in ether or chloroform.
iii. The pH of solution (1 → 200) of this product is 3.3~4.5.
iv. Melting point is 116~120°C
Confirmation test:
i. 0.1 g (0.05~0.14 g) of this product is dissolved with 30 mL of water. 0.5 mL of thissolution is added with 3 mL of p-dimethylaminobenzaldehyde-ferric chloride test
solution. The resulting solution is reddish violet.
ii. 0.01 g (0.005~0.014 g) of this product is dissolved with 2 mL of water and added with
1 drop of potassium permanganate test solution. The color of the test solution
disappears within 30 minutes.
iii. 5 mg (4.5~5.4 mg) of this product is added with 2 mL of citric acid-acetic anhydride
solution (0.5 → 100) and heated in a water bath, and then the solution is red to reddish
violet.
iv. 0.01 g (0.005~0.014 g) of this product is dissolved with 0.01 mol/L hydrochloric acidmethanol test solution to make 1,000 mL. This solution shows the absorption
maximum at the wavelengths of 322~327 nm by measuring the absorption spectrum.
v. 0.02 g (0.015~0.024 g) of this product is dissolved with 4 mL of water and added withdilute sodium hydroxide to be neutral. The resulting solution gives the qualitative
reaction iii. of citrate.
Purity test:
i. Clarity and color of solution: 0.5 g (0.45~0.54 g) of this product is dissolved with 10
mL of methanol. The resulting solution shall be yellow and clear.
ii. Chloride: 0.5 g (0.45~0.54 g) of this product is added with 40 mL of water anddissolved by heating. It is added with 6 mL of dilute nitric acid and water to make 50
mL. This is used as a sample solution. The chloride is tested using the control solution
prepared using 0.25 mL of 0.01 mol/L hydrochloric acid by the chloride test method.

555

Provisional Translation from Japanese Original

The opacity of the sample solution shall not be darker than that of the control solution(0.018 % or less).
iii. Sulfate: 0.5 g (0.45~0.54 g) of this product is added with 40 mL of water anddissolved by heating. It is added with 1 mL of dilute hydrochloric acid and water tomake 50 mL. This is used as a sample solution. The sulfate is tested using the control
solution prepared using 0.50 mL of 0.005 mol/L sulfuric acid by the sulfate test
method. The opacity of the sample solution shall not be darker than that of the control
solution (0.048 % or less).
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 3.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (30 mg/kg or
less).
v. Arsenic: 2.0 g (1.95~2.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (1 mg/kg or less).
vi. Cis isomer: 0.2 g (0.15~0.24 g) of this product is dissolved with methanol, transferred
to a 10 mL brown volumetric flask, and is further added with methanol to thegraduation line to make 10 mL. This is used as a sample solution. 1 mL of this solution
is measured using a volumetric pipette, transferred to a 100 mL brown volumetricflask, and added with methanol to the graduation line to make 100 mL. This is used asa control solution for cis isomer. Separately, 0.1 g (0.05~0.14 g) of anhydrous citricacid is weighed, dissolved with methanol, transferred to a 100 mL volumetric flask,
and is further added with methanol to the graduation line to make 100 mL. This is used
as a citric acid solution. 5 µL each of the sample, the control solution for cis isomer,
and the citric acid solution are spotted on a thin layer plate prepared using silica gel forthin-layer chromatography in the dark place. Then, they are developed toapproximately 10 cm using the upper layer of a mixed solution of methyl isobutyl
ketone, formic acid, and water (2:1:1) as the developing solvent in a dark place, andthe thin layer plate is dried at 100 °C for 15 minutes. The thin layer is placed in a bathfilled with iodine vapor, and then spots except those of morantel and citric acidobtained from the sample solution are not observed, or shall not be darker than thatobtained from the control solution for cis isomer (1 % or less).
Moisture: 3.5~5.0 % (0.5 g)

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Provisional Translation from Japanese Original

Ignition residue: 0.20 % or less (1 g)Assay: Approximately 0.1 g of this product is weighed to the digits of 0.001 g and the value
is recorded. It is dissolved with 0.01 mol/L hydrochloric acid-methanol test solution,transferred to a 200 mL brown volumetric flask, and is further added with the same test
solution to the graduation line to make 200 mL. This solution 2 mL is measured using a
volumetric pipette, transferred to a 100 mL brown volumetric flask, and added with 0.01
mol/L hydrochloric acid-methanol test solution to the graduation line to make 100 mL.
This is used as a sample solution. Separately, approximately 0.1 g of morantel citratereference standard is weighed to the digits of 0.001 g, and the value is recorded. It is
subjected to the same preparation procedure as that of the sample solution to prepare thestandard solution. The absorbances AT and AS of them at the wavelength 323 nm aremeasured, using 0.01 mol/L hydrochloric acid/methanol test solution as a control
solution.
Amount of Morantel citrate (C13 H16 N2 SꞏC6 H8 O7 ꞏH2O) (mg)
= Amount of Morantel citrate standard (mg) ×ATAS(c) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation(a) Compositional standards
This product is powder or particle in which the raw material for manufacturing of
Morantel citrate and fillers.
Content: This product is determined to contain Morantel citrate (C12H16N2S·C6H8O7·H2O)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.1 g of
the raw material for manufacturing of morantel citrate is weighed, added with 30 mL
of water, shaken for 5 minutes, and centrifuged. The supernatant 0.5 mL is added with
3 mL of p-dimethylaminobenzaldehyde-ferric chloride test solution. The resulting
solution is reddish violet.
ii. According to the amount of this product on the label, the amount containing 0.01 g of
the raw material for manufacturing of morantel citrate is weighed, added with 0.01
mol/L hydrochloric acid/methanol test solution, shaken and filtered. The filtrate shows
the absorption maximum at the wavelengths of 322~327 nm by measuring theabsorption spectrum.

557

Provisional Translation from Japanese Original

Assay: The amount of this product containing approximately 0.06 g of morantel citrate(C12H16N2S·C6H8O7·H2O) is weighed to three significant digits and the value is recorded.
It is placed in a brown volumetric flask, added with 150 mL of 0.01 mol/L hydrochloric
acid/methanol test solution, shaken for 30 minutes, added with 0.01 mol/L hydrochloric
acid/methanol test solution to accurately make 250 mL, and centrifuged. 5 mL of thesupernatant is measured using a volumetric pipette, transferred to a brown stoppered
flask, added with 5 mL of the solution, in which 0.12 g (0.115~0.124 g) of 2-hydroxy-mtoluic acid is dissolved with 100 mL of 0.01 mol/L hydrochloric acid/methanol test
solution, using a volumetric pipette. This is used as a sample solution. Separately,
approximately 0.06 g of morantel citrate reference standard is weighed to the digits of 0.1
mg, and the value is recorded. It is dissolved with 0.01 mol/L hydrochloric acid/methanol
test solution, transferred to a 250 mL volumetric flask, and added with 0.01 mol/Lhydrochloric acid/methanol test solution to the graduation line to make 250 mL. 5 mL ofthis solution is measured using a volumetric pipette and subjected to the same preparation
procedure as that of the sample solution to prepare the standard solution. 5 µL each of thesample and standard solutions are tested by the liquid chromatography under thefollowing conditions. Based on the obtained chromatograms, the peak heights of morantel
citrate and the internal standard solution of each solution are measured, and the ratio of
the peak height of morantel citrate to the peak height of the internal standard solution, HT
and HS are determined.
Amount of Morantel citrate (C12 H16 N2 SꞏC6 H8 O7 ꞏH2O) (mg)
= Amount of Morantel citrate standard (mg) ×HT
HSOperating conditions:
Detector: Ultraviolet absorptiometer (measurement wavelength: 320 nm,sensitivity :0.08AUFS)
Column: A stainless tube (inner diameter: 4.6 mm, length: 150 mm) is filled with 10 µm
of octadecyl silylation silica gel.
Column temperature: room temperature.
Mobile phase: Mixed solution of the solution, for which 1,000 mL of 0.05 mol/L
potassium dihydrogenphosphate is added with phosphoric acid (1 → 10) and adjusted
to pH 3.3, and acetonitrile (3:1).
Flow rate: 1.5 mL/min.
Column selection: A column to be used is selected when the standard solution 5 µL is
operated under the above conditions, morantel and the internal standard solution are
eluted in that order, and the separation degree is 20 or greater.

558

Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Morantel citrate
is applied mutatis mutandis.

(128) Nicarbazin
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried and determined, it contains 67.4~73.0 % of 4,4'dinitrocarbanilide (C13H10N4O5) and 27.7~30.0 % of 2-hydroxy-4, 6-dimethylpyrimidine(C6H8N2O).
Physical and chemical properties:
i. This product is yellow-brown to green-yellow powder without odor or with a slightly
specific odor.
ii. This product is hard to dissolve in dimethylformamide and is extremely hard todissolve in water, ethanol, ether, and chloroform.
iii. Melting point: Approximately 260 °C (dissolution)
Confirmation test:
i. The absolute ethanol solution of this product (1 → 15,000) 15 mL is added with 5 mLof sulfanilic acid test solution and 5 mL of newly prepared sodium nitrite solution (1→ 100), tightly stoppered, heated in a water bath at 65 °C for 10 minutes. The
resulting solution is red.
ii. The absolute ethanol solution (1 → 15,000) 15 mL is added with 5 mL of potassiumhydroxide/ethanol solution (1 → 100), and the resulting solution is yellow.
iii. The sample solution obtained by the assay of 4,4'-dinitrocarbanilide of the absorption
maximum is at the wavelengths of 428~432 nm by measuring the absorption spectrum.
iv. The sample solution obtained by the assay of 2-hydroxy-4, 6-dimethylpyrimidine of
the absorption maximum is at the wavelengths of 538~542 nm by measuring theabsorption spectrum.
Purity test:
i. pH: 0.20 g (0.195~0.204 g) of this product is weighed, added with 20 mL of water andstirred. The resulting solution shall be pH 5.0 to 7.0.
ii. Chloride: 0.20 g (0.195~0.204 g) of this product is weighed, added with 20 mL ofwater, boiled for 2 minutes, allowed to cool, and filtered. The filtrate 5 mL is measured
and added with 6 mL of dilute nitric acid and water to make 50 mL to prepare thesample solution. The chloride is tested using the control solution prepared using 0.4

559

Provisional Translation from Japanese Original

mL of 0.01 mol/L hydrochloric acid by the chloride test method. The opacity of thesample solution shall not be darker than that of the control solution (0.28 % or less).
iii. Sulfate: 0.20 g (0.195~0.204 g) of this product is weighed, added with 20 mL ofwater, boiled for 2 minutes, allowed to cool, and filtered. The filtrate 2 mL is measured
and added with 1 mL of dilute hydrochloric acid and water to make 50 mL to prepare
the sample solution. The sulfate is tested using the control solution prepared using 0.6
mL of 0.005 mol/L sulfuric acid by the sulfate test method. The opacity of the sample
solution shall not be darker than that of the control solution (1.44 % or less).
iv. Ammonium salt: 0.30 g (0.295~0.304 g) of this product is placed in a flask and addedwith 1 g (0.5~1.4 g) of magnesium oxide and 70 mL of water, and the flask isconnected to a distillation device. A 100 mL measuring cylinder with 2 mL of 0.1
mol/L hydrochloric acid is used as a receiver and the distillation is continued until 40
mL of solution is collected. The collected solution is added with water to make 100
mL and 10 mL of this solution is measured using a volumetric pipette, transferred to a
Nessler tube, added with water to make 40 mL, and added with 2 mL of sodiumhydroxide solution (1 → 10) and 2 mL of Nessler reagent. The color of the resulting
solution shall not be darker than that of the solution prepared by that 15 mL ofammonia standard solution in a Nessler tube added with water to make 40 mL
subjected to the same procedure for the sample (0.5 % or less).
v. Free 2-hydroxy-4, 6-dimethylpyrimidine: 0.5 g of this product is weighed to the digitsof 0.001 g and the value is recorded. It is mixed with 25 mL of pH 7.0 phosphatebuffer, shaken for 10 minutes, and filtered within 30 seconds. A few mL of the first
filtrate is removed and 5 mL of the clear filtrate is measured and added with pH 7.0
phosphate buffer to make 100 mL. When the absorbance AT of this solution at the
maximum wavelength near 295 nm within 30 seconds using water as a control
solution, the amount of free 2-hydroxy-4, 6-dimethylpyrimidine (C6H8N2O) shall be
3.0 % or less.
Amount of 4,4'-dinitrocarbanilide (C13 H10 N4 O5 ) (mg) =AT
× 1,000
118

Loss on drying: 1.0 % or less (1 g, reduced pressure, 110 °C, 1 hour)
Ignition residue: 0.3% or less (1 g)Assay: This product is dried and approximately 0.05 g of it is weighed to the digits of 0.1
mg and the value is recorded. It is added with dimethylformamide, dissolved by heating,allowed to cool, transferred to a 100 mL volumetric flak, is further added withdimethylformamide to the graduation line to make 100 mL, mixed well. This is used as asample stock solution. Separately, nicarbazin reference standard is dried in the same way

560

Provisional Translation from Japanese Originalas the sample, and approximately 0.05 g of it is weighed to the digits of 0.1 mg and thevalue is recorded. It is added with dimethylformamide, dissolved by heating, allowed tocool, transferred to a 100 mL volumetric flak, is further added with dimethylformamide
to the graduation line to make 100 mL. This is used as a standard stock solution.
4,4'-dinitrocarbanilide: 4 mL of the sample stock solution is measured using a volumetricpipette, transferred to a 200 mL volumetric flask, added with ethanol to make 200 mL,
and mixed well. This solution 15 mL is measured using a volumetric pipette, transferred
to a 25 mL volumetric flask, added with 5 mL of potassium hydroxide/ethanol solution (1→ 100), and is further added with ethanol to the graduation line to make 25 mL. This isused as a sample solution. The standard stock solution 4 mL is measured using a
volumetric pipette, transferred to a 200 mL volumetric flask, added with ethanol to make200 mL, and hereinafter subjected to the same procedure as the sample solution toprepare the standard solution. When the absorbances AS and AT of the standard andsample solutions at the maximum wavelength near 430 nm are measured using potassiumhydroxide/ethanol solution (1 → 500) as a control solution.
Amount of 4,4'-dinitrocarbanilide (C13 H10N4 O5 ) (mg)
= Nicarbazin reference standard (mg) ×AT
× 0.7089AS2-hydroxy-4, 6-dimethylpyrimidine: 4 mL of the sample stock solution is measured using a
volumetric pipette, transferred to a 200 mL volumetric flask, added with a mixed solutionof dimethylformamide and ethanol (1:1) to the graduation line to make 200 mL, andmixed well. 10 mL of this solution is measured using a volumetric pipette, transferred toa 50 mL stoppered test tube, added with 10 mL of newly prepared sulfanilamide test
solution and mixed. It is added with 2 mL of newly prepared sodium nitrite solution (1 →50), tightly stoppered, mixed, heated in a water bath at 65 °C for 15 minutes, cooled withrunning water, allowed to stand at room temperature for 20 minutes. This is used as asample solution. Separately, 4 mL of the standard stock solution is measured using a
volumetric pipette, transferred to a 200 mL volumetric flask, added with ethanol to thegraduation line to make 200 mL, and from here is subjected to the same procedure as the
sample solution to prepare the standard solution. Separately, 10 mL of a mixed solutionof dimethylformamide and ethanol (1:1) is measured with a volumetric pipette,
transferred to a 50 mL stoppered test tube, and hereinafter subjected to the sameprocedure as the sample solution to prepare the blank test solution. The absorbances AS
and AT of the standard and sample solutions at the maximum wavelength near 540 nm aremeasured using the blank test solution as a control solution.
Amount of 2-hydroxy-4,6-dimethylpyrimidine (C6 H8 N2 O) (mg)561

Provisional Translation from Japanese Original= Nicarbazin reference standard (mg) ×AT× 0.2912
AS(b) Standard of storage method
It shall be stored in a lightproof airtight container.
B. Preparation(a) Compositional standards
This product is solid and powder in which the raw material for manufacturing of
nicarbazin and fillers.
Content: This product is determined to contain nicarbazin (C19H18N6O6) corresponding to98~106 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing 5 mg of
the raw material for manufacturing of nicarbazin is weighed, added with 60 mL ofabsolute ethanol and shaken well while heating. It is allowed to cool and filtered, andthe filtrate 15 mL is added with 5 mL of sulfanilic acid test solution and 5 mL of
newly prepared sodium nitrite solution (1 → 100), tightly stoppered, and heated in awater bath at 65 °C for 10 minutes. The resulting solution is red.
ii. According to the amount of this product on the label, the amount containing 5 mg of
the raw material for manufacturing of nicarbazin is weighed, added with 60 mL ofabsolute ethanol and shaken well while heating. It is allowed to cool and filtered, andthe filtrate 15 mL is added with 5 mL of potassium hydroxide/ethanol solution (1 →100). The resulting solution is yellow.
iii. In the measurement of the absorption spectrum of the sample solution prepared by theassay for 4,4'-dinitrocarbanilide, the absorption maximum is at the wavelengths of428~432 nm.
iv. In the measurement of the absorption spectrum of the sample solution prepared by theassay for 2-hydroxy-4, 6-dimethylpyrimidine, the absorption maximum is at the
wavelengths of 538~542 nm.
Assay: The amount of this product containing approximately 0.125 g of nicarbazin(C19H18N6O6) is weighed to three significant digits and the value is recorded. It is placedin a 200 mL volumetric flask, added with 150 mL of dimethylformamide, heated in awater bath for 15 minutes, and shaken for 15 minutes. It is allowed cool, added withdimethylformamide to the graduation line to make 200 mL, mixed well and centrifuged
for 5 minutes. This supernatant is collected to use as the sample stock solution.
Separately, nicarbazin reference standard is dried, and approximately 0.05 g of it isweighed to the digits of 0.01 mg and the value is recorded. It is added with562

Provisional Translation from Japanese Original

dimethylformamide, dissolved by heating, allowed to cool, transferred to a 100 mL
volumetric flask, added with dimethylformamide to the graduation line to 100 mL. This isused as a standard stock solution.
4,4'-dinitrocarbanilide: The sample stock solution 4 mL is measured using a volumetricpipette, transferred to a 250 mL volumetric flask, added with ethanol to make 250 mL. 15
mL of this solution is measured using a volumetric pipette, transferred to a 25 mL
volumetric flask, added with 5 mL of potassium hydroxide/ethanol solution (1 → 100)
using a volumetric pipette, and added with ethanol to the graduation line to make 25 mL,
and mixed well. This is used as a sample solution. Separately, 5 mL of the standard stock
solution is measured using a volumetric pipette, transferred to a 250 mL volumetric flask,
and added with ethanol to the graduation line to make 250 mL. 15 mL of this solution ismeasured using a volumetric pipette, and from here subjected to the same procedure asthat for the sample solution to prepare the standard solution. The absorbances AT and ASof the sample and standard solutions at the maximum wavelength near 430 nm aremeasured using potassium hydroxide/ethanol solution (1 → 500) as a control solution.
Amount of nicarbazin (C19 H18 N6 O6 ) (mg)
= Nicarbazin reference standard (mg) ×

AT
× 2.5
AS

2-hydroxy-4, 6-dimethylpyrimidine: 4 mL of the sample stock solution is measured using a
volumetric pipette, transferred to a 250 mL volumetric flask, added with a mixed solutionof dimethylformamide and ethanol (1:1) to the graduation line to make 250 mL. 10 mL ofthis solution is measured using a volumetric pipette, transferred to a 50 mL stoppered test
tube, added with 10 mL of newly prepared sulfanilamide test solution and mixed. It is
added with 2 mL of newly prepared sodium nitrite solution (1 → 50), tightly stoppered,
mixed, heated in a water bath at 65 °C for 15 minutes, cooled with running water,allowed to stand for 20 minutes. This is used as a sample solution. Separately, 5 mL of
the standard stock solution is measured using a volumetric pipette, transferred to a 250
mL volumetric flask, and added with a mixed solution of dimethylformamide and ethanol(1:1) to the graduation line to make 250 mL, and from here subjected to the sameprocedure as that for the sample solution to prepare a standard solution. Separately, 10
mL of a mixed solution of dimethylformamide and ethanol (1:1) is subjected to the sameprocedure as that for the sample solution to prepare a blank test solution. The absorbances
AS and AT of the standard and sample solutions at the maximum wavelength near 540 nm
are measured using the blank test solution as a control solution.
Amount of nicarbazin (C19 H18 N6 O6 ) (mg)563

Provisional Translation from Japanese Original= Nicarbazin reference standard (mg) ×

AT
× 2.5
AS

(b) Standard of storage method
It shall be stored in a lightproof capped container.

(129) Calcium Halofuginone Polystyrene-sulfonate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried and determined, it contains 95.0 % or more ofcalcium halofuginone polystyrene-sulfonate (C16H17BrClN3O3·Calcium halofuginone
Polystyrene sulfonate).
Physical and chemical properties: This product is pale yellow-brown to brown powder withlittle odor.
Confirmation test:
i. 0.2 g (0.15~0.24 g) of this product is weighed, added with 20 mL of 6 mol/Lhydrochloric acid test solution, stirred for 30 minutes, and centrifuged. 10 mL of thesupernatant is measured and added with 10 mL of methanol to prepare a sample
solution. Separately, 20 mg (19.5~20.4 mg) of halofuginone hydrobromide is weighed
and dissolved with 100 mL of a mixed solution of 6 mol/L hydrochloric acid test
solution and methanol (1:1) to prepare a standard solution. 10 µL each of the sample
and standard solutions are measured, and spotted on a thin layer plate prepared using
silica gel for thin-layer chromatography (with a fluorescent agent). Then, they aredeveloped to approximately 15 cm with the developing solvent, a mixed solution ofchloroform, methanol and ammonia solution (90:10:1), and the thin layer plate is air
dried. When it is irradiated with ultraviolet light (dominant wavelength: 254 nm), thespots obtained from the sample and standard solutions are dark purple and their Rfvalues are equal.
ii. 0.2 g (0.15~0.24 g) of this product is weighed, added with 5 mL of 6 mol/Lhydrochloric acid test solution, stirred for 5 minutes, filtered, and neutralized withammonia test solution. The resulting solution gives the qualitative reaction iii. of
calcium salt.
iii. 20 mg (19.5~20.4 mg) of this product is weighed, added with 5 mL of 6 mol/Lhydrochloric acid test solution, stirred for 5 minutes, and centrifuged, and thesupernatant is removed. Then, the residue is added with 10 mL of water, shaken, andcentrifuged, and the supernatant is removed. This residue is added to 2 mL of copper

564

Provisional Translation from Japanese Original

sulfate solution (1 → 10), shaken well, and centrifuged, and the supernatant isremoved. Then, this residue is added with 10 mL of water, shaken, and centrifuged,
and the supernatant is removed. This residue is added with 2 mL of ammonia test
solution and shaken for 1 minute, and the resulting solution is dark blue.
iv. 0.5 g (0.45~0.54 g) of this product is weighed, added with 10 mL of 6 mol/Lhydrochloric acid test solution, stirred for 15 minutes, and filtered. In the measurementof the absorption spectrum of the solution of 1 mL of the filtrate and 25 mL of water,
the absorption maximum is at the wavelengths of 241~245 nm.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (20 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Halofuginone: This product is dried, and 0.5 g (0.45~0.54 g) of it is weighed, addedwith 50 mL of dimethyl sulfoxide using a volumetric pipette, stirred for 30 minutes,
and centrifuged. 1 mL of this supernatant is measured using a volumetric pipette,
transferred to a 50 mL volumetric flask, added with hydrochloric acid (1 → 100) to thegraduation line to make 50 mL. This is used as a sample solution. Separately, 17 mg(16.5~17.4 mg) of halofuginone hydrobromide is weighed, dissolved withhydrochloric acid (1 → 100), transferred to a 500 mL volumetric flak, and is further
added with hydrochloric acid (1 → 100) to the graduation line to make 500 mL. The
solution 1 mL is measured using a volumetric pipette, transferred to a 200 mL
volumetric flask, added with hydrochloric acid (1 → 100) to the graduation line to
make 200 mL. This is used as a standard solution. When 100 µL each of the sample
and standard solutions are measured, and tested by the liquid chromatography under
the following conditions, the peak area of halofuginone in the sample solution shall beequal to or lower than that of halofuginone in the standard solution (0.1 % or less).
Operating conditions:
Detector: Ultraviolet absorptiometer (measurement wavelength: 243 nm)

565

Provisional Translation from Japanese Original

Column: A stainless tube (inner diameter: 4~6 mm, length: 250 mm) is filled withapproximately 5 µm of octadecyl silylation silica gel.
Column temperature: Constant temperature at around 40 °C
Mobile phase: Ammonium acetate 4.2 g (4.15~4.24 g) is dissolved with approximately 200mL of water, added with 6.4 mL of acetic acid, and added with water to make 1,000 mL.
This solution 700 mL is measured and added with 300 mL of acetonitrile.
Flow rate: 1.5 mL/min.
iv. Cis isomer: 17 mg (16.5~17.4 mg) of Halofuginone hydrobromide (cis isomer) is
weighed, dissolved with hydrochloric acid (1 → 100), transferred to a 500 mL
volumetric flask, and is further added with hydrochloric acid (1 → 100) to thegraduation line to make 500 mL. 1 mL of this solution is measured using a volumetricpipette, transferred to a 500 mL volumetric flask, and added with 500 mL ofhydrochloric acid (1 → 100) to the graduation line to prepare the standard solution.
When 100 µL each of the sample and standard solutions obtained by the assay aremeasured and tested by the liquid chromatography under the same conditions as iii.,
the peak area of halofuginone in the sample solution shall be equal to or lower thanthat of halofuginone in the standard solution (0.2 % or less).
Loss on drying: 6.0 % or less (1 g, 105 °C, 4 hours)
Ignition residue: After drying, 29.0 % or less (1 g)Polystyrene sulfonate: This product is dried, 0.5 g of it is weighed to the digits of 0.1 mg,
and the value is recorded. It is added with 50 mL of 6 mol/L hydrochloric acid test
solution, shaken for 15 minutes, and centrifuged, and the supernatant is removed. This
procedure is repeated four times. The residue is added with 40 mL of water and shaken.
The washing is repeated until opacity does not occur even if silver nitrate test solution isadded in the washings, and the residue is dried at 105 °C for 12 hours. The amount of theresidue weighed to the digits of 0.1 mg shall be 87.0 % or less.
Amount of polystyrene sulfonate (%) =Amount after drying (mg)× 100Collected amount of this product (mg)Assay: This product is dried and approximately 0.5 g of it is weighed to the digits of 0.001
g, and the valued is recorded. It is added with approximately 10 mL of 6 mol/Lhydrochloric acid test solution, dispersed, washed with 6 mol/L hydrochloric acid test
solution into a chromatographic tube with glass wool at the bottom (inner diameter 10mm, height 150 mm). A 250 mL volumetric flask is placed as a receiver under thechromatographic tube. Then, it is eluted using 6 mol/L hydrochloric acid test solution
until the flow rate is approximately 225 mL. Then it is added with 6 mol/L hydrochloric
acid test solution to the graduation line to make 250 mL. 1 mL of the solution is measured

566

Provisional Translation from Japanese Originalusing a volumetric pipette, transferred to a 50 mL volumetric flask, added with water tothe graduation line to make 50 mL. This is used as a sample solution. The absorbance Aof the sample solution at the maximum wavelength near 243 nm is measured usinghydrochloric acid (1 → 100) as a control solution.
Amount of Calcium halofuginone Polystyrene sulfonate (C16H17BrClN3O3) (mg)=A
× 125,000
86.3(b) Standard of storage method
It shall be stored in an airtight container.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Calcium
Polystyrene sulfonate and fillers.
Content: When this product is determined, it contains 80 µg/mg or less or the amountcorresponding to 90~110 % of the amount on the label of halofuginone calcium
polystyrene-sulfonate (C16H17BrClN3O3·Calcium halofuginonePolystyrene sulfonate)
Confirmation test:i. According to the amount of this product on the label, the amount containing 0.02 g of
the raw material for manufacturing of calcium halofuginone polystyrene-sulfonate isweighed, added with 50 mL of 6 mol/L hydrochloric acid test solution, stirred forapproximately 15 minutes, and filtered. Approximately 2 mL of porous styrene
divinylbenzene copolymer refined resin immersed in methanol is washed twice withwater, and it is added in the above filtrate, stirred for 15 minutes, filtered with a glass
filter. This resin is washed twice with 20 mL of water, added with 5 mL of methanol,
stirred, and filtered. This methanol solution is used as a sample solution. Separately,
0.02 g (0.015~0.024 g) of the raw material for manufacturing of calcium halofuginone
polystyrene-sulfonate is weighed, and subjected to the same procedure to prepare thesolution. This is used as a standard solution. 10 µL each of the sample and standard
solutions are weighed, and the confirmation test i. of the raw material for
manufacturing of calcium halofuginone polystyrene-sulfonate is hereinafter appliedmutatis mutandis.
ii. The sample solution obtained by the assay of calcium halofuginone polystyrene
sulfonate of the absorption maximum is at the wavelengths of 243~247 nm bymeasuring the absorption spectrum.
Assay: The amount of this product containing 0.05 g of the raw material for manufacturingof calcium halofuginone polystyrene-sulfonate is weighed to three significant digits and567

Provisional Translation from Japanese Original

the value is recorded. It is gradually added with 20 mL of 6 mol/L hydrochloric acid test
solution and shaken well. When the bubble release stops, it is washed with a little 6 mol/Lhydrochloric acid test solution into a chromatographic tube with glass wool at the bottom(inner diameter 10 mm, height 150 mm). A 200 mL volumetric flask is placed as areceiver under the chromatographic tube. Then, it is eluted using 6 mol/L hydrochloric
acid test solution until the flow rate is approximately 180 mL. Then it is added with 6
mol/L hydrochloric acid test solution to the graduation line to make 200 mL. 10 mL ofthis solution is measured using a volumetric pipette, transferred to a 50 mL volumetricflask, added with water to the graduation line to make 50 mL. This is used as a sample
solution. The absorbance A of the sample solution at the maximum wavelength near 245
nm is measured using hydrochloric acid (10 → 100) as a control solution.

Amount of Calcium halofuginone Polystyrene sulfonate (C16H17BrClN3O3) (mg)=A
× 10,000
78.5(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Calcium
halofuginone polystyrene sulfonate is applied mutatis mutandis.
(c) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“使用上の注意 この飼料添加物は、鶏に過剰投与した場合発育障害が起こるので、定められた添加量を厳守するとともに、均一に配合するよう注意すること。”
Precautions: Overdose of this feed additive causes developmental disorder in chicken, so
strictly follow the specified amount to be added and make sure to mix it uniformly.

(130) Saccharin Sodium
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is dried at 120 °C for 4 hours and then determined, it contains
99.0 % or greater saccharin sodium (C7H4NNaO3S).
Physical and chemical properties: This product is colorless to white crystals, white
crystalline powder or white powder with an extremely sweet taste. Even a 10,000-foldsolution is sweet.

568

Provisional Translation from Japanese OriginalConfirmation test:
i. 0.5 g (0.45~0.54 g) of this product is dissolved with 10 mL of water, added with 1 mLof dilute hydrochloric acid, allowed to stand for 1 hour, and the white crystalline
precipitation is collected by filtration, washed well, dried at 105 °C for 2 hours, andthe melting point is 226~230 °C.
ii. 20 mg (19.5~20.4 mg) of this product is mixed with 40 mg (39.5~40.4 mg) of resorcin,
added with 10 drops of sulfuric acid, and gently heated. When the mixture turns darkgreen, it is allowed to cool and dissolved with 10 mL of water and 10 mL of 1 mol/L
sodium hydroxide test solution. The resulting solution emits green fluorescence.
iii. 0.1 g (0.05~0.14 g) of this product is dissolved with 5 mL of 1 mol/L sodiumhydroxide test solution, gently heated, evaporated to dryness, then melted avoiding
carbonization, and allowed to cool when ammonia odor is not produced. The residue isdissolved with approximately 20 mL of water, neutralized with dilute hydrochloric
acid, and filtered. The filtrate with 1 drop of ferric chloride test solution is purple toreddish violet.
iv. The residue obtained by ashing of this product gives the qualitative reaction of sodium
salt.
Purity test:
i. Clarity and color of solution: This product is powdered, and 1 g (0.5~1.4 g) each of thepowder is dissolved in 1.5 mL of water and in 70 mL of ethanol. These solutions shallbe colorless and clear.
ii. Free acid and free alkali: 1 g (0.5~1.4 g) of this product is dissolved in 10 mL of newly
boiled and cooled, added with 1 drop of phenolphthalein test solution. The resulting
solution shall not be red. Furthermore, the solution added with an additional 1 drop of
0.1 mol/L sodium hydroxide shall be red.
iii. Benzoate and salicylate: 0.5 g (0.45~0.54 g) of this product is dissolved in 10 mL ofwater and added with 5 drops of acetic acid and 3 drops of ferric chloride test solution.
In the resulting solution there shall be no precipitation. Also, the solution shall not be
purple to reddish violet.
iv. Heavy metal: 1.0 g (0.95~1.04 g) of this product is dissolved in 45 mL of water andadded with 2 mL of diluted acetic acid. The solution is used as a sample solution to
perform the test on heavy metal, the amount of heavy metal shall be 10 mg/kg or less.
v. Arsenic: 2.5 g (2.45~2.54 g) of this product is placed in a dissolution flask, added with
10 mL of nitric acid and 5 mL of sulfuric acid and heated. When the solution remains
brown, it is allowed to cool, added with an additional 1 mL of nitric acid and heated.
The procedure is repeated until the solution becomes colorless to pale yellow, and then569

Provisional Translation from Japanese Original

the solution is heated until white smoke emerges. It is allowed to cool, added with 10
mL of water and 15 mL of saturated ammonium oxalate solution and again heateduntil white smoke emerges. It is allowed to cool and added with 25 mL of water, and 5
mL of this solution is measured and used as a sample solution. When the sample
solution is tested on arsenic by the method using device A, the color of absorbing
solution shall not be darker than the standard color. However, the standard color ismade by the procedure so that 10 mL of arsenic standard solution is transferred to adissolution flask, added with 10 mL of nitric acid and 5 mL of sulfuric acid andhereinafter subjected to the same procedure as that for the sample solution (4 mg/kg or
less).
vi. o-toluenesulfonamide: 10 g (9.5~10.4 g) of this product is dissolved in 50 mL ofwater and extracted three times with 30 mL each of ethyl acetate. The ethyl acetate
layer is collected, washed with 30 mL of 25 w/v% sodium chloride solution, anddehydrated with anhydrous sodium sulfate, ethyl acetate is distilled away, and theresidue is dissolved with 5 mL of internal standard solution . This is a sample solution.
Separately, o-Toluenesulfonamide-ethyl acetate solution (1 → 1,000) 1 mL is
measured, heated in a water bath to remove ethyl acetate, and the residue is dissolved
with 5 mL of internal standard solution to prepare a standard solution. When thesample and standard solutions are tested by the gas chromatography under thefollowing conditions, the ratio H/HS of the peak height of caffeine (HS) and otoluenesulfonamide (H) in the sample solution shall not exceed the ratio H'/H'S of thepeak height of caffeine (H'S) and o-toluenesulfonamide (H') in the standard solution(100 mg/kg or less). However, as the internal standard solution solution, caffeine-ethyl
acetate solution (1 → 5,000) is used.
Operating conditions:
Column packings: Diatomaceous earth carrier for gas chromatograph (177~250 µm) is
added with chloroform solution containing succinate diethylene glycol polyester,corresponding to 3 % of such carrier to evaporate chloroform, and dried.
Column tube: Glass or stainless tube with inner diameter of 3~4 mm and length of 1 m
Column temperature: Constant temperature at around 195~205 °C
Detector: Hydrogen flame ionization detector
Carrier gas: Nitrogen gas is used. The column temperature and the flow of carrier gas areadjusted so that caffeine appears approximately 6 minutes after.
Loss on drying: 15 % or less (1 g, 120 °C, 4 hours)
Assay: This product is dried at 120 °C for 4 hours and approximately 0.3 g of it is weighted
to the digits of 0.001 g, and the value is recorded. It is dissolved with 20 mL of glacial

570

Provisional Translation from Japanese Original

acetic acid for nonaqueous titration, and titrated with 0.1 mol/L perchloric acid (indicator:
2 drops of crystal violet-glacial acetic acid test solution). The end point of titration is thetime when the color of the solution changes from purple through blue to green.
Separately, a blank test is performed in the same way and corrections are made.
1 mL of 0.1 mol/L perchloric acid = 20.52 mg C7H4NNaO3S(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of saccharin sodium
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of saccharin
sodium is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or crystalline powder of a mixture of the raw material for
manufacturing of saccharin sodium and fillers.
Content: This product is determined to contain saccharin sodium (C7H4NNaO3S)
corresponding to 90~110 % of the amount on the label.
Confirmation test:i. According to the amount of this product on the label, the amount containing
approximately 0.5 g of the raw material for manufacturing of saccharin sodium isweighed, added with 30 mL of water, shaken well, and if necessary filtered. Thissolution is added with 1 mL of dilute hydrochloric acid and allowed to stand for 1
hour, and the resulting white crystalline precipitation is filtered, washed well, anddried at 105 °C for 2 hours. This is used as a sample. The sample 0.5 g (0.45~0.54 g) is
weighed, and the confirmation test i. of the raw material for manufacturing ofsaccharin sodium is hereinafter applied mutatis mutandis.
ii. 20 mg (19.5~20.4 mg) of the sample i. is weighed, and the confirmation test ii. of raw
material for manufacturing of saccharin sodium is hereinafter applied mutatis
mutandis.
iii. 0.1 g (0.05~0.14 g) of the sample i. is dissolved in 5 mL of 1 mol/L sodium hydroxide
test solution, gently heated, evaporated to dryness, and allowed to cool. The residue isdissolved in approximately 20 mL of water, neutralized with dilute hydrochloric acid,571

Provisional Translation from Japanese Original

and filtered. The filtrate added with 1 drop of ferric chloride test solution is purple toreddish violet.
iv. For the sample of i., the confirmation test iv. of the raw material for manufacturing ofsaccharin sodium id applied mutatis mutandis.
Assay: The amount of this product containing approximately 0.3 g of saccharin sodium(C7H4NNaO3S) is weighed to three significant digits and the value is recorded. It isdissolved with 20 mL of glacial acetic acid for nonaqueous titration and titrated with 0.1
mol/L perchloric acid (indicator: 2 drops of crystal violet-glacial acetic acid test solution).
In this case, the end point of titration is the time when the color of the solution changes
from purple to blue. A blank test is performed in the same manner, and corrections are
made.
1 mL of 0.1 mol/L perchloric acid = 20.52 mg C7H4NNaO3S(b) Standard of storage method
It shall be stored in a capped container.

(131) Flavour
A. Raw material for manufacturing(a) Compositional standards
This product is liquid including one or not less than two of the following active
ingredients: esters, ethers, ketone, fatty acids, aliphatic higher alcohols, aliphatic higher
aldehydes, aliphatic higher hydrocarbons, terpene hydrocarbons, phenol ethers, phenol,
aromatic alcohols, aromatic aldehydes, and lactone.
(b) Standard of storage method
It shall be stored in a lightproof capped container.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of raw materials for manufacturing are applied mutatis
mutandis. However, no adverse effects are observed in the amount used for feed.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing is applied mutatis
mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is powder or particles of a mixture of the raw material for manufacturing
and fillers. No adverse effects are observed in the amount used for feed.

572

Provisional Translation from Japanese Original(b) Standard of storage method
The standard of storage method of the raw material for manufacturing is applied mutatis
mutandis.
D. Preparation (Part 3 oil liquid)(a) Compositional standards
This product is oil liquid of a mixture of the raw material for manufacturing andhydrogenated oils, higher saturated fatty acids, fatty acids, vegetable oil, or animal fats. No
adverse effects are observed in the amount used for feed.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing is applied mutatis
mutandis.

(132) Amylase
Amylase (Part 1)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 3,500 starch saccharification power units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.0~7.5.
iii. This product produces maximum enzyme activity at pH 4.5~5.5.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.

573

Provisional Translation from Japanese Original

Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b)Standard of manufacturing method
For manufacturing, the amylase producing strain of Aspergillus oryzae or Rhizopus
delemar is cultured, after the cultivation, the culture is filtered, or extracted with water, andthen filtered to remove bacterial cells, and then the filtrate is dried or the precipitate
produced by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Amylase (Part 1)
are applied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Amylase(Part 1) are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Amylase (Part1) is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Amylase (Part 1) isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces or powder of the mixture of the raw material for manufacturing of s
Amylase (Part 1) and fillers.
Enzymatic activity unit: This product contains 85~170 % of starch saccharification power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Amylase (Part1) is applied mutatis mutandis.

574

Provisional Translation from Japanese Original(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Amylase (Part 1) isapplied mutatis mutandis.
Amylase (Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 2,000 starch saccharification power units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is dark brown liquid with a specific odor.
ii. pH: The pH of this product is 5.0~7.5.
iii. This product has maximum enzyme activity at pH 5.0~7.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less)
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Ignition residue: After drying, 20.0 % or less (1 g)
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b) Standard of manufacturing method
For manufacturing: The amylase producing strain of Bacillus subtilis or Bacillus
amyloliquefaciens is cultured, after the cultivation, the culture is filtered, or extracted withwater, then filtered to remove bacterial cells, and then the filtrate is concentrated.
(c) Standard of storage method
It shall be stored in a capped container and in the freezer.

575

Provisional Translation from Japanese Original(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Amylase (Part 1) isapplied mutatis mutandis.
B. Preparation (Part 1)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturing
of of s Amylase (Part 2) and fillers.
Enzymatic activity unit: This product contains 85~170 % of starch saccharification power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b) Standard of storage method
It shall be stored in a capped container.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Amylase (Part 1) isapplied mutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is semiliquid, oil liquid, or water-soluble liquid of the mixture of the raw
material for manufacturing of amylase (Part 2) and hydrated silicon dioxide, hydrated
amorphous silicon oxide, silicic acid, calcium silicate, light anhydrous silicic acid,hydrogenated oils, higher saturated fatty acids, glycerin, fatty acids, salt, vegetable oil,animal fats, lactose, maltose, white sugar, glucose, silicic anhydride or anhydrous tricalcium
silicates.
Enzymatic activity unit: This product contains 85~170 % of starch saccharification power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b) Standard of storage method
The standards of storage method of amylase (Part 2) Preparation (Part 1) is appliedmutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Amylase (Part 1) isapplied mutatis mutandis.

576

Provisional Translation from Japanese Original(133) Alkaline ProteaseAlkaline protease (Part 1)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 100,000 protein digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is white to pale brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.5~8.5.
iii. This product produces maximum enzyme activity at pH 7.0~9.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b)Standard of manufacturing method
For manufacturing, alkaline protease producing strain of Aspergillus melleus, Bacillus
licheniformis, or Streptomyces caespitosus is cultured, after the cultivation, the culture isfiltered, or extracted with water and filtered to remove bacterial cells, and then the filtrate isdried or the precipitate produced by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.

577

Provisional Translation from Japanese Original(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Alkaline protease(Part 1) are applied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Alkaline
protease (Part 1) are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Alkaline
protease (Part 1) is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 1) is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Alkaline protease (Part 1) is mixed with calcium sulfate as appropriate, and sodium
sulfate and fillers.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Alkaline
protease (Part 1) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 1) is applied mutatis mutandis.
D. Preparation (Part 3 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing of
Alkaline protease (Part 1) and glycerin.

578

Provisional Translation from Japanese Original

Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Alkaline
protease (Part 1) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 1) is applied mutatis mutandis.
Alkaline Protease (Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 80,000 protein digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown liquid with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.5~8.5.
iii. This product produces maximum enzyme activity at pH 7.0~9.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Ignition residue: After drying, 10.0% or less (1 g)
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.

579

Provisional Translation from Japanese Original(b)Standard of manufacturing method
For manufacturing, alkaline protease producing strain of Bacillus subtilis is cultured, after
the cultivation, the culture is filtered or extracted with water and filtered to remove bacterial
cells, and then the filtrate is concentrated.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Alkaline protease (Part 2) is mixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the protein digestive power test method.
(b) Standard of storage method
It shall be stored in a lightproof capped container.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 2) is applied mutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing of
Alkaline protease (Part 2) and propylene glycol.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the protein digestive power test method.
(b) Standard of storage method
It shall be stored in a capped container and under 25 degrees.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 2) is applied mutatis mutandis.

580

Provisional Translation from Japanese Original

Alkaline protease (Part 3)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 200,000 protein digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown to rich brown liquid with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.0~7.0.
iii. This product produces maximum enzyme activity at pH 9.0~11.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Ignition residue: After drying, 5.0 % or less (1 g)
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b)Standard of manufacturing method
For manufacturing, alkaline protease producing recombinant, whose host is the strain
belonging to Bacillus subtilis is cultured, after the cultivation, the culture is filtered or
extracted with water and filtered to remove bacterial cells, and then the filtrate isconcentrated.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.

581

Provisional Translation from Japanese Original

B. Preparation (Part 1)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Alkaline protease (Part 3) is mixed with sodium sulfate as appropriate, and sucrose andfillers.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Alkaline
protease (Part 3) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 3) is applied mutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is water-soluble liquid in which the raw material for manufacturing of
Alkaline protease (Part 3) is mixed with sodium benzoate and potassium sorbate as
appropriate, and sorbitol, glycerin.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 in the protein digestive capacity test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Alkaline
protease (Part 3) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Alkaline protease(Part 3) is applied mutatis mutandis.

(134) Xylanase
A. Raw material for manufacturing(a) Compositional standards

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Provisional Translation from Japanese Original

Enzymatic activity unit: This product contains 4,000 xylan saccharification power units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown liquid with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.0~7.0.
iii. This product produces maximum enzyme activity at pH 5.0~6.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Ignition residue: After drying, 10.0 % or less (1 g)
Enzymatic activity test: It is tested by the xylan saccharification power test method.
(b)Standard of manufacturing method
For manufacturing, Xylanase producing strain of Trichoderma longibrachiatum iscultured, after the cultivation, the culture is filtered or extracted with water and filtered toremove bacterial cells, and then the filtrate is concentrated.
(c) Standard of storage method
It shall be stored in a lightproof and airtight container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Xylanase is mixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of xylan saccharification power
units on the label by an enzymatic activity test.

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Provisional Translation from Japanese Original

Enzymatic activity test: It is tested by the xylan saccharification power test method.
(b) Standard of storage method
It shall be stored in a lightproof capped container.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of xylanase is appliedmutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing of
xylanase and D-sorbitol.
Enzymatic activity unit: This product contains 85~170 % of starch saccharification power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the starch saccharification power test method.
(b) Standard of storage method
It shall be stored in a capped container and 25 degrees or less.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of xylanase is appliedmutatis mutandis.

(135) Xylanase/Pectinase complexing enzyme
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 400 xylan saccharification power units ormore and 12,000 pectin saccharification power units or more per 1 g by an enzymatic
activity test.
Physical and chemical properties:
i. This product is pale yellow and pale brown pawder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.0~7.0.
iii.This product has xylanase maximum enzyme activity at pH 3.0~5.0 and pectinasemaximum enzyme activity at pH 3.5~4.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is placed in a quartz or porcelain pot,loosely covered with a lid, and weakly heated to be carbonized. It is allowed to cool,
added with 2 mL of nitric acid and 5 drops of sulfuric acid, heated carefully until whitesmoke emerges, and ignited at 500~600 °C to be ashed. It is allowed to cool, added584

Provisional Translation from Japanese Original

with 2 mL of hydrochloric acid, and evaporated to dryness in a water bath, and theresides is moisturized with 3 drops of hydrochloric acid, added with 10 mL of boilingwater, and heated for 2 minutes. Then, it is adjusted pH 5.0~6.0 with ammonia test
solution, added with 2 mL of diluted acetic acid, if necessary filtered, and washed with
10 mL of water. The filtrate and the washings are transferred to a Nessler tube andadded with 50 mL of water. This is used as a sample solution. By the heavy metals test
using a control solution prepared with 5.0 mL of lead standard solution by method No.
2 of the heavy metals test method, the color of the sample solution shall not be darker
than that of the control solution (50 mg/kg or less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 30 % or less (1 g)
Enzymatic activity test: It is tested by the xylan saccharification power test method and thepectin saccharification power test method.
(b)Standard of manufacturing method
For manufacturing, xylanase-pectinase complexing enzyme producing strain of
Aspergillus usamii mut. shiro-usamii is cultured, after the cultivation, the culture is
extracted with water and filtered to remove bacterial cells, and then the precipitate produced
by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation(a) Compositional standards
This product is powder of the mixture of the raw material for manufacturing of
Xylanase/pectinase complexing enzyme and fillers.
Enzymatic activity unit: This product contains 85~200 % of xylan saccharification power
units and pectin saccharification power units on the label by an enzymatic activity test.

585

Provisional Translation from Japanese Original

Enzymatic activity test: It is tested by the Xylane saccharification power test method andpectin saccharification power test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of
Xylanase/pectinase complexing enzyme is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Xylanase/pectinase
complexing enzyme is applied mutatis mutandis.

(136) β-glucanase
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 4,000 beta-glucan saccharification power
units or more/g by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown liquid with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 3.5~6.0.
iii. This product has maximum enzyme activity at pH 3.5~5.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Ignition residue: 10.0 % or less (1 g)
Enzymatic activity test: It is tested by the beta-glucan saccharification power test method.
(b)Standard of manufacturing method586

Provisional Translation from Japanese Original

For manufacturing, β-glucanase producing strain of Trichoderma longibrachiatum iscultured, after the cultivation, the culture is filtered or extracted with water and filtered toremove bacterial cells, and then the filtrate is concentrated.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof β-glucanase complexing enzyme and fillers.
Enzymatic activity unit: This product contains 85~170 % of β-glucanase saccharification
power units and pectin saccharification power units on the label by an enzymatic activity
test.
Enzymatic activity test: It is tested by the β-glucanase saccharification power test method.
(b) Standard of storage method
It shall be stored in a lightproof capped container.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of β-glucanase
complexing enzyme is applied mutatis mutandis.
C. Preparation (Part 2 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing of
β-glucanase and water.
Enzymatic activity unit: This product contains 85~170 % of β-glucanase saccharification
power units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the β-glucanase saccharification power test method.
(b) Standard of storage method
It shall be stored in a lightproof capped container.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of β-glucanase is appliedmutatis mutandis.

587

Provisional Translation from Japanese Original(137) Acid Protease
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 60,000 protein digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale white to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 3.5~7.0.
iii. This product has maximum enzyme activity at pH 2.0~4.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 15.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by method No. 2 of the protein digestive power test
method.
(b)Standard of manufacturing method
For manufacturing, acid protease producing strain of Aspergillus niger, Aspergillus saito,
Rhizopus delemar, or Rhizopus niveus is cultured, after the cultivation, the culture is filteredor extracted with water and filtered to remove bacterial cells, and then the filtrate is dried or
the precipitate produced by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.

588

Provisional Translation from Japanese Original

B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Acid protease areapplied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Acid
protease are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Acid protease isapplied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Acid protease isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of Acid
protease complexing enzyme and fillers.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
and pectin saccharification power units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 2 of the protein digestive power test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Acid protease isapplied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Acid protease isapplied mutatis mutandis.

(138) Cellulase
Cellulase(Part 1)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 1,500 fiber disruption capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:

589

Provisional Translation from Japanese Originali. This product is pale yellow to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.0~7.5.
iii. This product produces maximum enzyme activity at pH 4.0~5.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by the fiber disruption capacity test method.
(b)Standard of manufacturing method
For manufacturing, cellulase producing strain of Trichoderma reesei, or Trichoderma
viride is cultured, after the cultivation, the culture is filtered or extracted with water andfiltered to remove bacterial cells, and then the filtrate is dried or the precipitate produced byadding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Cellulase (Part 1)
are applied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of
Cellulase (Part 1) are applied mutatis mutandis.

590

Provisional Translation from Japanese Original(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cellulase (Part1) is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Cellulase (Part 1) isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Cellulase (Part 1) is mixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of fiber disruption power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by the fiber disruption capacity test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cellulase (Part1) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Cellulase (Part 1) isapplied mutatis mutandis.
Cellulase (Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 250 fiber saccharification power units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.0~7.5.
iii. This product produces maximum enzyme activity at pH 4.0~5.5.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).

591

Provisional Translation from Japanese Original

ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by the fiber saccharification power test method.
(b)Standard of manufacturing method
For manufacturing, Cellulase producing strain of Acremonium cellulolyticus, Aspergillus
aculeatus, Humicola insolens, or Trichoderma viride is cultured, after the cultivation, theculture is filtered or extracted with water and filtered to remove bacterial cells, and then thefiltrate is dried or the precipitate produced by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Cellulase (Part 2)
are applied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of
Cellulase (Part 2) are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cellulase (Part2) is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Cellulase (Part 2) isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards

592

Provisional Translation from Japanese Original

This is pieces, powder or particles of the granulated mixture with the raw material for
manufacturing Cellulase (Part 2) and as appropriate polyvinyl alcohol solution, or of the
mixture added with sodium sulfate and fillers.
Enzymatic activity unit: This product contains 85~170 % fiber saccharification power
capacity units/g or more by an enzymatic activity test.
Enzymatic activity test: It is tested by the fiber saccharification power test method.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of
Cellulase (Part 2) are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cellulase (Part2) is applied mutatis mutandis.
D. Preparation (Part 3 liquid)(a) Compositional standards
This product is water-soluble liquid of the mixture of the raw material for manufacturingof Cellulase (Part 2) and, as appropriate sorbitol, glycerin, salt, glucose, lactose, maltose,
and white sugar.
Enzymatic activity unit: This product contains 85~170 % fiber saccharification power
capacity units/g or more by an enzymatic activity test.
Enzymatic activity test: It is tested by the fiber saccharification power test method.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of
Cellulase (Part 2) are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Cellulase(Part2) is applied mutatis mutandis.

(139) Cellulase/Protease/Pectinase Complexing enzyme
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 800 fiber disruption capacity units or
more, 300 protein digestive capacity units or more and 200 pectin liquefaction capacity
units or more per 1 g by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown to yellow brown powder with a slightly specific odor.

593

Provisional Translation from Japanese Original

ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.0~7.0.
iii. This product has cellulose, protease and pectinase maximum enzyme activities at pH
3.5~5.0, pH 3.0~4.0 and pH 4.5~5.5, respectively.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by the fiber disruption capacity test method, method No.
2 of the protein digestive capacity test method and the pectin liquefaction capacity test
method.
(b)Standard of manufacturing method
For manufacturing, cellulase/protease/pectinase complexing enzyme producing strain of
Irpex lacteus is cultured, after the cultivation, the culture is filtered or extracted with waterand filtered to remove bacterial cells, and then the filtrate is dried or the precipitate
produced by adding a solvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity of eachcellulose, cellulose, and pectinase shall be described on the immediate container or theimmediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme are applied mutatis mutandis.

594

Provisional Translation from Japanese Original(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This is pieces, powder or particles of the mixture with the raw material for manufacturing
Cellulase/protease/pectinase complexing enzyme and fillers.
Enzymatic activity unit: This product contains 85~200 % of fiber disruption power units,
protein digestive power units and pectin liquefaction power units on the label by an
enzymatic activity test.
Enzymatic activity test: It is tested by the fiber disruption capacity test method, method No.
2 of the protein digestive capacity test method and the pectin liquefaction capacity test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of
Cellulase/protease/pectinase complexing enzyme is applied mutatis mutandis.

(140) Neutral Protease
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 60,000 protein digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.5~7.5.
iii. This product produces maximum enzyme activity at pH 6.0~7.0.

595

Provisional Translation from Japanese Original

Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by method No. 3 of the protein digestive capacity test
method.
(b)Standard of manufacturing method
For manufacturing, Neutral protease enzyme producing strain of Aspergillus oryzac iscultured, after the cultivation, the culture is filtered or extracted with water and filtered toremove bacterial cells, and then the filtrate is dried or the precipitate produced by adding asolvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Neutral protease
are applied mutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Neutral
protease are applied mutatis mutandis.

596

Provisional Translation from Japanese Original(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Neutral protease
is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Neutral protease isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Neutral Protease is mixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of protein digestive power units
on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 3 of the protein digestive power test
method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Neutral protease
is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Neutral protease isapplied mutatis mutandis.

(141) Phytase
Phytase (Part 1)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 1,500 phytic acid degradation capacity
units/g or more by an enzymatic activity test.
Physical and chemical properties:
i. This product is white to pale brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 4.5~7.5.
iii. This product produces maximum enzyme activity at pH 5.0~6.0.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample

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Provisional Translation from Japanese Original

solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is placed in a dissolution flask, addedwith 10 mL of nitric acid and 5 mL of sulfuric acid, and gently heated. When thesolution remains brown, it is allowed to cool, added with an additional 1~2 mL ofnitric acid and heated. The procedure is repeated until the solution becomes colorlessto slightly yellow. After cooling, it is added with 0.5 mL of perchloric acid and heateduntil white smoke emerges. After cooling, it is added with 15 mL of saturated
ammonium oxalate and again heated until white smoke emerges. It is allowed to cooland added with water to make approximately 10 mL. This is used as a sample solution.
When the sample solution is tested on arsenic by the method using device A, the colorof absorbing solution shall not be darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 12.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (0.5 g)
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method.
(b) Standard of manufacturing method
For manufacturing, Neutral protease enzyme producing strain of Aspergillus niger iscultured, after the cultivation, the culture is filtered or extracted with water and filtered toremove bacterial cells, and then the filtrate is dried or the precipitate produced by adding asolvent in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Phytase (Part 1) areapplied mutatis mutandis.
(b) Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Phytase(Part 1) are applied mutatis mutandis.

598

Provisional Translation from Japanese Original(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase (Part 1)
is applied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Phytase (Part 1) isapplied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This is pieces, powder or particles of the mixture with the raw material for manufacturing
Phytase (Part 1) and fillers.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase (Part 1)
is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Phytase (Part 1) isapplied mutatis mutandis.
Phytase ((1) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 phytic acid degradation capacity
units/g or more by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown liquid with a slightly specific odor.
ii. The solution (1 → 100) of this product is pH 3.5~6.5.
iii. This product produces maximum enzyme activity at pH 4.0~6.0.
Purity test: The purity test of the raw material for manufacturing of Phytase (Part 1) areapplied mutatis mutandis.
Ignition residue: 5.0 % or less (0.5 g)
Enzymatic activity test: It is tested by method No. 2 of the phytic acid degradation capacity
test method.

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Provisional Translation from Japanese Original

Preparation of sample solution: Polysorbate 20 0.1 g (0.05~0.14 g) is dissolved with 0.25
mol/L acetic acid/hydrochloric acid buffer, and is further added with the same buffer tomake 1 liter. This is used as a diluent. The amount required for testing of the sample isweighed to three significant digits and the value is recorded. It is added with the diluent
to make its concentration 50 phytic acid degradation capacity units/mL, vigorously stirred
to be dissolved. This is used as a sample stock solution. Adequate volume of this stock
solution is measured using a volumetric pipette, added with the diluent to make its
concentration 0.5 phytic acid degradation capacity units/mL to prepare a sample solution.
(b) Standard of manufacturing method
For manufacturing, the phytase producing recombinant, whose host is the strain
belonging to Aspergillus oryzae, is cultured, after the cultivation, the culture is filtered or
extracted with water and filtered to remove bacterial cells and then the filtrate isconcentrated.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing ofphytase ((1) of part 2), as appropriate sorbitol, and glycerin.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 2 of the phytic acid digestive power test
method.
Preparation of sample solution: The preparation of sample solution of the raw material for
manufacturing of phytase ((1) of part 2) is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((1) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Phytase ((1) of Part 2)
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards

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Provisional Translation from Japanese Original

This product is pieces or particles of a mixture of the raw material for manufacturing ofphytase ((1) of part 2), as appropriate sodium sulfate, and fillers.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 2 of the phytic acid digestive power test
method.
Preparation of sample solution: 5 g (4.5~5.4 g) of polysorbate 20 and 0.6 g (0.55~0.64 g) ofbovine serum albumin is dissolved with 0.25 mol/L acetic acid/hydrochloric acid buffer,and is further added with the same buffer to make 1 liter. This is used as an extract. For
the diluent, the raw material for manufacturing of phytase ((1) of part 2) is appliedmutatis mutandis. The amount required for testing of the sample is weighed to threesignificant digits and the value is recorded. It is added with the extract to make its
concentration of 50 phytic acid degradation capacity units/mL, and sonicated for 20
minutes using a triangular rotor while vigorously stirring, is further vigorously stirred for
20 minutes to be dissolved, and then centrifuged for 3 minutes at 14,000 rpm. The
supernatant is used as a sample stock solution. The adequate volume of the stock solution
is measured using a volumetric pipette, and added with the diluent to make its
concentration of 0.25 phytic acid degradation capacity units/mL. This is used as a sample
solution.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((1) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Phytase ((1) of Part 2)
is applied mutatis mutandis.
Phytase ((2) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 phytic acid degradation capacity
units/g or more by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow brown liquid with a slightly specific odor.
ii. The solution (1 → 100) of this product is pH 3.5~6.5.
iii. This product produces maximum enzyme activity at pH 5.0~6.0.

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Provisional Translation from Japanese Original

Purity test: The purity test of the raw material for manufacturing of Phytase (Part 1) areapplied mutatis mutandis.
Ignition residue: 5.0 % or less (0.5 g)
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “0.005 mol/L acetic acid/sodium
acetate buffer adjusted to pH showing maximum enzyme activity of the sample” shall be
replaced by the words “buffer containing 0.1 g of polysorbate 20 per 1 liter of 0.005
mol/L acetic acid/sodium acetate adjusted to pH showing maximum enzyme activity ofthe sample”.
(b) Standard of manufacturing method
For manufacturing, the phytase producing recombinant, whose host is the strain
belonging to Aspergillus niger, is cultured, and after the cultivation bacterial cells are
disinfected and filtered, or extracted with water and filtered to be removed, and then thefiltrate is concentrated.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is water-soluble liquid of a mixture of the raw material for manufacturing ofphytase ((2) of part 2), as appropriate sorbitol.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “0.005 mol/L acetic acid/sodium
acetate buffer adjusted to pH showing maximum enzyme activity of the sample” shall be
replaced by the words “buffer containing 0.1 g of polysorbate 20 per 1 liter of 0.005
mol/L acetic acid/sodium acetate adjusted to pH showing maximum enzyme activity ofthe sample”.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((2) ofpart 2) is applied mutatis mutandis.

602

Provisional Translation from Japanese Original(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity and thefollowing words shall be described on the immediate container or the immediate wrapper ofthis product.
Valid period: 6 months from the day manufacture
C. Preparation (Part 2)(a) Compositional standards
This product is pieces or particles of a granulated mixture of the raw material for
manufacturing of phytase ((2) of part 2) and corn starch and magnesium sulfate.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “0.005 mol/L acetic acid/sodium
acetate buffer adjusted to pH showing maximum enzyme activity of the sample” shall be
replaced by the words “buffer containing 0.1 g of polysorbate 20 per 1 liter of 0.005
mol/L acetic acid/sodium acetate adjusted to pH showing maximum enzyme activity ofthe sample”.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((2) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity and thefollowing words shall be described on the immediate container or the immediate wrapper ofthis product.
Valid period: 9 months from the day manufacture
Phytase ((3) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 phytic acid degradation capacity
units/g or more by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow brown liquid with a slight characteristic odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 3.5~6.5.
iii. This product produces maximum enzyme activity at pH 4.5~6.0.

603

Provisional Translation from Japanese Original

Purity test: The purity test of the raw material for manufacturing of Phytase (Part 1) areapplied mutatis mutandis.
Ignition residue: 5.0 % or less (0.5 g)
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “0.005 mol/L acetic acid/sodium
acetate buffer adjusted to pH showing maximum enzyme activity of the sample” shall be
replaced by the words “buffer containing 0.1 g of polysorbate 20 per 1 liter of 0.2 mol/Lacetic acid/sodium acetate adjusted to pH showing maximum enzyme activity of thesample”.
(b) Standard of manufacturing method
Shall be manufactured by culturing phytase production recombinants hosted by bacterial
strains belonging to Schizosaccharomyces pombe, filtering the culture product ,or filtrating
the culture product after extracting with water, to remove bacterial cells when the culturing
is complete, and by concentrating the filtrate.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is a water-soluble liquid in which sodium chloride, citric acid and sorbitol
are mixed into the raw material for manufacturing of Phytase ((3) of part 2).
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “0.005 mol/L acetic acid/sodium
acetate buffer adjusted to pH showing maximum enzyme activity of the sample” shall be
replaced by the words “buffer containing 0.1 g of polysorbate 20 per 1 liter of 0.2 mol/Lacetic acid/sodium acetate adjusted to pH showing maximum enzyme activity of thesample”.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((3) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label604

Provisional Translation from Japanese OriginalThe Standards of the label of the raw material for manufacturing of Phytase ((3) of Part 2)
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This is pieces, powder or particles of the granulated mixture with the raw material for
manufacturing Phytase ((3) of Part 2) and as appropriate polyvinyl alcohol solution, andthen of the mixture added and mixed or granulated with sodium sulfate and fillers, or citricacid, wheat flour and calcium propionate are added into.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method No. 1 of the phytic acid degradation capacity
test method. However, the words in the Procedures “add and melt 0.005 mol/L acetic
acid/sodium acetate buffer adjusted to pH showing maximum enzyme activity of thesample” shall be replaced by the words “add buffer containing 0.1 g of polysorbate 20 per
1 liter of 0.2 mol/L acetic acid/sodium acetate adjusted to pH showing maximum enzyme
activity of the sample and stir in ice for 60 minutes to dissolve”.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((3) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Phytase ((3) of Part 2)
is applied mutatis mutandis.
Phytase ((4) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 phytic acid degradation capacity
units/g or more by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale brown liquid.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 3.5~6.5.
iii. This product produces maximum enzyme activity at pH 3.5~4.5.
Purity test:i. Lead: Weigh 0.5 g (0.45~0.54 g) of this product and when a test for lead is performedby the limit test for lead (atomic absorption spectrometry No.1), the amount shall be
no more than 20 μg/g.

605Provisional Translation from Japanese Original

ii. Arsenic: Apply mutatis mutandis the purity test ii. For source material for
manufacturing Phytase (Part 1).
iii. Antibacterial activity: Apply mutatis mutandis the purity test iii for source material
for manufacturing Phytase (Part 1).
Ignition residue: After drying, 5.0 % or less (0.5 g)
Enzymatic activity test: It is tested by method 1 of Phytic acid decomposition power test
method. However, in the section of “Preparation of substrate solution”, “dissolve byadding 50 mL of 0.2 mol/L acetate-sodium acetate buffer solution adjusted to pH
showing the maximum enzymatic activity of the sample, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution” shall read as “dissolve by adding 50 mL of 0.2 mol/Lacetate-sodium acetate buffer solution adjusted to pH 5.5, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution” Also, in the section of “Procedure”, “Weigh up to threesignificant digits of the amount of sample required to perform the test,record the number,dissolve by adding 0.005 mol/L acetate-sodium acetate buffer adjusted topH showing the
maximum enzymatic activity of the sample so that the concentration per 1mL becomes
0.04~0.06 phytic acid decomposition activity units. If necessary, perform filtration, use
this solution as the sample solution” shall read as “To 1.0 g (0.995~1.004 g) of a sample,
add a buffer which contains polysorbate 20 at a rate of 0.1 g per 1 L of 0.2-mol/L acetic
acid-sodium acetate buffer whose pH is adjusted for the sample to show the maximum
enzyme activity to dissolve, filter if necessary, and dilute using the same buffer so thatthe phytate degradation capacity unit per mL becomes 0.04~0.06 to prepare the sample
solution.”(b) Standard of manufacturing method
Shall be manufactured by culturing phytase production recombinants hosted by bacterial
strains belonging to Trichoderma reesei, filtering the culture product (or filtrating theculture product after extracting with water) to remove bacterial cells when the culturing iscomplete, and by concentrating the filtrate.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
C. Preparation (Part 1 Liquid)(a) Compositional standards
This product is aqueous liquid in which sodium chloride and sorbitol are mixed into raw
material for manufacturing phytase ((4) of Part 2).

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Provisional Translation from Japanese Original

Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation
capacity units on the label by an enzymatic activity test.
Enzymatic activity test: It is tested by method 1 of the Phytic acid decomposition power test
method. However, in the section of “Preparation of substrate solution”, “dissolve byadding 50 mL of 0.2 mol/L acetate-sodium acetate buffer solution adjusted to pH
showing the maximum enzymatic activity of the sample, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution” shall read as “dissolve by adding 50 mL of 0.2 mol/Lacetate-sodium acetate buffer solution adjusted to pH 5.5, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution.” Also, in the section of “Procedure”, “Weigh up to threesignificant digits of the amount of sample required to perform the test, record the number,dissolve by adding 0.005 mol/L acetate-sodium acetate buffer adjusted to pH showing the
maximum enzymatic activity of the sample so that the concentration per 1 mL becomes
0.04~0.06 phytic acid decomposition activity units. If necessary, perform filtration, use
this solution as the sample solution.” shall read as “To 1.0 g (0.995~1.004 g) of a sample,
add a buffer which contains polysorbate 20 at a rate of 0.1 g per 1 L of 0.2 mol/L acetic
acid-sodium acetate buffer whose pH is adjusted for the sample to show the maximum
enzyme activity to dissolve, filter if necessary, and dilute using the same buffer so thatthe phytate degradation capacity unit per mL becomes 0.04~0.06 to prepare the sample
solution.”(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((4) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
C. Preparation (Part 2)(a) Compositional standards
This product is small piece, powder or granule in which an aqueous solution where
polyvinyl alcohol, sodium phytate, sodium monohydrogen phosphate, sodium dihydrogen
phosphate, potassium monohydrogen phosphate, potassium dihydrogen phosphate, inositol,
vegetable fats and oils, white sugar, and starch selected as necessary are mixed into andadded to raw material for manufacturing Phytase ((4) of Part 2), sodium sulfate and carrier
are further added if necessary, and mixed or granulated.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation
capacity units on the label by an enzymatic activity test.

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Provisional Translation from Japanese Original

Enzymatic activity test: It is tested by method 1 of the Phytic acid decomposition power test
method. However, in the section of “Preparation of substrate solution”, “dissolve byadding 50 mL of 0.2 mol/L acetate-sodium acetate buffer solution adjusted to pH
showing the maximum enzymatic activity of the sample, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution” shall read as “dissolve by adding 50 mL of 0.2 mol/Lacetate-sodium acetate buffer solution adjusted to pH 5.5, adjust to pH 5.5 by adding 0.2
mol/L acetic acid test solution” Also, in the section of “Procedure”, “Weigh up to threesignificant digits of the amount of sample required to perform the test,record the number,dissolve by adding 0.005 mol/L acetate-sodium acetate buffer adjusted topH showing the
maximum enzymatic activity of the sample so that the concentration per 1mL becomes
0.04~0.06 phytic acid decomposition activity units. If necessary, perform filtration, use
this solution as the sample solution.” shall read as “To 1.0 g (0.995~1.004 g) of a sample,
add a buffer which contains polysorbate 20 at a rate of 0.1 g per 1 L of 0.2-mol/L acetic
acid-sodium acetate buffer whose pH is adjusted for the sample to show the maximum
enzyme activity and stir in ice for 60 minutes to dissolve, filter if necessary, and diluteusing the same buffer so that the phytate degrading power unit per mL becomes
0.04~0.06 to prepare the sample solution.”(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((4) ofpart 2) is applied mutatis mutandis.
(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
Phytase ((5) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 phytic acid degradation capacity
units/g or more using an enzymatic activity test.
Physical and chemical properties:
i. This product is yellow brown liquid.
ii. The pH of a solution of this product in water (1 → 100) is 3.5 to 6.0.
iii. This product produces maximum enzyme activity at pH 3.5~5.5.
Purity test:608

Provisional Translation from Japanese Originali. Lead: 2.0 g (1.95~2.04 g) of this product is weighed. When lead is tested using the leadtest method (Method No. 1 of the atomic absorption spectrophotometry), the amountof lead shall be 5 μg/g or less.
ii. Arsenic: The purity test ii. of the raw material for manufacturing of Phytase (Part 1) isapplied mutatis mutandis.
iii. Antibacterial activity: The purity test iii. of the raw material for manufacturing of
Phytase (Part 1) is applied mutatis mutandis.
Ignition residue: 5.0 % or less (0.5 g)
Enzymatic activity test: It is tested using method No. 2 of the phytic acid degradation
capacity test method.
Preparation of sample solution: The preparation of sample solution of the raw material for
manufacturing of Phytase ((1) of Part 2) is applied mutatis mutandis.
(b) Standard of manufacturing method
For manufacturing, the Phytase producing recombinant, whose host is the strain
belonging to Aspergillus niger, is cultured, and after the cultivation bacterial cells are
disinfected and filtered to be removed, the filtrate is concentrated.
(c) Standard of storage method
It shall be stored in a lightproof sealed container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is a water-soluble liquid in which fillers are mixed into the raw material for
manufacturing of Phytase ((5) of Part 2).
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label using an enzymatic activity test.
Enzymatic activity test: It is tested using method No. 2 of the phytic acid degradation
capacity test method.
Preparation of sample solution: The preparation of sample solution of the raw material for
manufacturing of Phytase ((1) of Part 2) is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((5) ofPart 2) is applied mutatis mutandis.
(c) Standards of the label

609

Provisional Translation from Japanese Original

The standards of the label of the raw material for manufacturing of Phytase ((5) of Part 2)
is applied mutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is granules, pieces, or particles in which fillers and an aqueous solution of
polyvinyl alcohol are mixed into the raw material for manufacturing of Phytase ((5) of Part2), which is granulated, dried, and coated with oxidized polyethylene wax, oleic acid, andammonium hydroxide.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label using an enzymatic activity test.
Enzymatic activity test: It is tested using method No. 2 of the phytic acid degradation
capacity test method.
Preparation of sample solution: The preparation of sample solution of the raw material for
manufacturing of Phytase ((1) of Part 2) is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((5) ofPart 2) is applied mutatis mutandis.
(c) Standards of the label
The standards of the label of the raw material for manufacturing of Phytase ((5) of Part 2)
is applied mutatis mutandis.
Phytase ((6) of Part 2)
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 200,000 phytic acid degradation capacity
units/g or more using an enzymatic activity test.
Physical and chemical properties:
i. This product is yellow to brown powder or particles.
ii. The pH of a solution or suspension of this product in water (1 → 100) is 4.0 to 5.0.
iii. This product produces maximum enzyme activity at pH 2.5~5.0.
Purity test:i. Lead: 1.0 g (0.95~1.04 g) of this product is weighed. When lead is tested using the leadtest method (Method No. 1 of the atomic absorption spectrophotometry), the amountof lead shall be 5 μg/g or less.
ii. Arsenic: The purity test ii. of the raw material for manufacturing of Phytase (Part 1) isapplied mutatis mutandis.

610

Provisional Translation from Japanese Originaliii. Antibacterial activity: The purity test iii. of the raw material for manufacturing of
Phytase (Part 1) is applied mutatis mutandis.
Loss on drying: 12.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: 25.0 % or less (0.5 g)
Enzymatic activity test: It is tested using method No. 3 of the phytic acid degradation
capacity test method.
Preparation of sample solution: 2.00 g (1.995~2.004 g) of this product is weighed in a 100mL volumetric flask, and the value is recorded. To which 0.2 mol/L citric acid-sodium
citrate buffer (pH 5.5) is added up to the capacity mark to make 100 mL. This solution isstirred at room temperature for 1 hour, and centrifuged at 3,400×g for 10 minutes. The
supernatant is used as a sample stock solution. An appropriate amount of the sample
stock solution is measured using a volumetric pipette, and added with the said buffer tomake its concentration 0.1 phytic acid degradation capacity units/mL. This solution isused as a sample solution.
(b) Standard of manufacturing method
For manufacturing, the Phytase producing recombinant, whose host is the strain
belonging to Komagataella pastoris, is cultured, and after the cultivation the culture isfiltered to remove bacterial cells, the filtrate is spray dried.
(c) Standard of storage method
It shall be stored in a lightproof sealed container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
B. Preparation (Part 1 liquid)(a) Compositional standards
This product is a water-soluble liquid in which an aqueous sucrose solution is mixed into
the raw material for manufacturing of Phytase ((6) of Part 2).
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label using an enzymatic activity test.
Enzymatic activity test: It is tested using method No. 3 of the phytic acid degradation
capacity test method.
Preparation of sample solution: 5 mL of this product is measured using a volumetric pipette,
put in a 50 mL volumetric flask, and added with 0.2 mol/L citric acid-sodium citratebuffer (pH 5.5) up to the capacity mark to make 50 mL. This is used as a sample stock
solution. An appropriate amount of the sample stock solution is measured using a611

Provisional Translation from Japanese Original

volumetric pipette, and added with the said buffer to make its concentration 0.1 phyticacid degradation capacity units/mL. This solution is used as a sample solution.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((6) ofPart 2) is applied mutatis mutandis.
(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces, powder or particles in which wheat flour is added to the raw
material for manufacturing of Phytase ((6) of Part 2), mixed with an aqueous solution of αstarch, granulated, dried, and mixed with fillers as appropriate.
Enzymatic activity unit: This product contains 85~170 % of phytic acid degradation power
units on the label using an enzymatic activity test.
Enzymatic activity test: It is tested using method No. 3 of the phytic acid degradation
capacity test method.
Preparation of sample solution: The enzymatic activity test of the raw material for
manufacturing of Phytase ((6) of Part 2) is applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Phytase ((6) ofPart 2) is applied mutatis mutandis.
(c) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product.
(142) Muramidase
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: Perform the enzymatic activity test: 1 g of muramidase contains notless than 30,000 peptidoglycan units.
Physical and chemical properties
i. Muramidase is a light brown to deep brown liquid.

ii. The pH of an aqueous solution of muramidase or a suspension of muramidase inwater (1 → 100) is between 3.0 ~ 5.0.
iii. Muramidase has maximum enzymatic activity at a pH of between 4.0 ~ 7.5.
612

Provisional Translation from Japanese Original

Purityi. Lead: Perform the test for lead with 0.5 g (0.45 ~ 0.54 g) of muramidase as directedunder Lead Test (Method 1 of Atomic Absorption Spectrophotometry): the amount oflead shall be not more than 20 μg/g.

ii. Arsenic: Weigh 1.0 g (0.95 ~ 1.04 g) of muramidase, prepare the sample solution
according to Method 3 of the Arsenic Test, and perform the test for arsenic by themethod using Apparatus A: the color of the absorbing liquid shall have no more color
than the standard color (not more than 2 μg/g).

iii. Antibacterial activity: Perform the test with 1 g (0.5 ~1.4 g) of muramidase as
directed under Antibacterial Activity Assay for Micrococcus luteus ATCC 9341 andEscherichia coli ATCC 27166: the result should show no antibacterial activity.
Residue on ignition: 10.0 % or less (1 g)
Enzymatic activity test: Perform the test as directed under the Peptidoglycan Degradation
Test.
(b)Standard of manufacturing method
Culture a muramidase-producing recombinant using a strain belonging to
Trichodermareesei as a host. After culturing, filter the culture or filter upon extractingwith water to remove bacterial bodies, and concentrate the filtrate to produce
muramidase.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product .
B. Preparation (Part 1)(a) Compositional standards
Muramidase is a small piece, powder, or particle obtained by adding sodium sulfate asnecessary to the active ingredients for muramidase production and mixing the excipient.
Enzymatic activity unit: Perform the enzymatic activity test: muramidase contains not lessthan 85 % and not more than 170 % of the labeled peptidoglycan units.
Enzymatic activity test: Perform the test as directed under the Peptidoglycan Degradation
Test.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Muramidase isapplied mutatis mutandis.
(c) Standards of the label

613

Provisional Translation from Japanese OriginalThe Standards of the label of the raw material for manufacturing of Muramidase isapplied mutatis mutandis.
C. Preparation (Part 1)(a) Compositional standards
Muramidase is a water-soluble liquid obtained by mixing sorbitol with the active ingredientsfor muramidase production.
Enzymatic activity unit: Perform the enzymatic activity test: muramidase contains not lessthan 85 % and not more than 170 % of the labeled peptidoglycan units.
Enzymatic activity test: Perform the test as directed under the Peptidoglycan Degradation
Test.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Muramidase isapplied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Muramidase isapplied mutatis mutandis.

(143) Lactase
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 25,000 lactase units/g or more by an
enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow to brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.5~7.5.
iii. This product produces maximum enzyme activity at pH 4.0~5.5.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by614

Provisional Translation from Japanese Original

the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 30.0 % or less (1 g)
Enzymatic activity test: It is tested by the lactase test method.
(b)Standard of manufacturing method
For manufacturing, Lactase enzyme producing strain of Aspergillus oryzac is cultured,
after the cultivation, the culture is filtered or extracted with water and filtered to remove
bacterial cells, and then the filtrate is dried or the precipitate produced by adding a solventin the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this productB. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Lactase are appliedmutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Lactase
are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Lactase isapplied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Lactase is appliedmutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces, powder, or particles in which the raw material for manufacturingof Lactase is mixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of Lactase units on the label by an
enzymatic activity test.

615

Provisional Translation from Japanese Original

Enzymatic activity test: It is tested by Lactase test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Lactase isapplied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Lactase is appliedmutatis mutandis.

(144) Lipase
A. Raw material for manufacturing(a) Compositional standards
Enzymatic activity unit: This product contains 5,000 lipid digestive capacity units/g ormore by an enzymatic activity test.
Physical and chemical properties:
i. This product is pale yellow to pale brown powder with a slightly specific odor.
ii. The solution or aqueous suspension (1 → 100) of this product is pH 5.0~6.5.
iii. This product produces maximum enzyme activity at pH 6.0~7.5.
Purity test:
i. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 5.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (50 mg/kg or
less).
ii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
iii. Antibacterial activity: When 1 g (0.5~1.4 g) of this product is weighed and tested by
the antibacterial activity test method, it shall not exhibit antibacterial activity.
Loss on drying: 10.0 % or less (1 g, 105 °C, 3 hours)
Ignition residue: After drying, 25.0 % or less (1 g)
Enzymatic activity test: It is tested by the lipid digestive capacity test method.
(b)Standard of manufacturing method

616

Provisional Translation from Japanese Original

For manufacturing, the lipase producing strain, Alcaligenes eutrophus, Candida
cylindracea or Rhizopus japonicus is cultured, and after the cultivation, the culture is
extracted with weak alkaline solution and filtered to be removed, and then the filtrate isdried, or the precipitate produced by adding a solvent or salt in the filtrate is dried.
(c) Standard of storage method
It shall be stored in a lightproof capped container.
(d) Standards of the label
The pH value (to one place of decimals) showing maximum enzyme activity shall be
described on the immediate container or the immediate wrapper of this product .
B. Preparation (Part 1)(a) Compositional standards
The compositional standards of the raw material for manufacturing of Lipase are appliedmutatis mutandis.
(b)Standard of manufacturing method
The Standard of manufacturing method of the raw material for manufacturing of Lipase
are applied mutatis mutandis.
(c) Standard of storage method
The standard of storage method of the raw material for manufacturing of Lipase isapplied mutatis mutandis.
(d) Standards of the label
The Standards of the label of the raw material for manufacturing of Lipase is appliedmutatis mutandis.
C. Preparation (Part 2)(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of Lipase ismixed with fillers.
Enzymatic activity unit: This product contains 85~170 % of lipid digestive power units on
the label by an enzymatic activity test.
Enzymatic activity test: It is tested by lipid digestive power test method.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of Lipase isapplied mutatis mutandis.
(c) Standards of the label
The Standards of the label of the raw material for manufacturing of Lipase is appliedmutatis mutandis.

617

Provisional Translation from Japanese Original(145) Enterococcus faecalis
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-driedEnterococcus faecalis NT strain.
Origin: The original strain is Enterococcus faecalis NT strain which was separated fromhuman feces in 1983.
Physical and chemical properties: This product is a facultative anaerobic Gram-positive
coccus. It breaks down lactose to produce lactate, breaks down melezitose, but does notbreak down arabinose or melibiose.
Confirmation test:
i. This product is applied on medium No. 3, cultured at 36~38 °C for 2 days. The colony
forming on the medium is round, raised, with smooth surface, and milky.
ii. This product is applied on medium No. 3 and cultured at 36~38 °C for 2 days. A
platinum-loop amount of water is placed on a slide glass. The colony is placed on theslide glass using a platinum wire, stirred to be suspended, spread to the proper size,and dried at room temperature or by fire from a distance. Then, it is passed through theflame 2~3 times to be fixed. This is used as a sample. In the test of the sample byGram stain, cocci stained blue-violet to dark purple are observed.
iii. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days. In the test using the colony forming on themedium by the lactic acid producing capacity test method, production of lactic acid isobserved.
iv. Medium No. 9 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured or aerobically at 36~38 °C for 2 days. The growth of thebacteria is observed under both conditions.
v. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days. The results of the test on arabinose, melibiose,and melezitose using the colony forming on the medium by method No. 2 of theglycolytic capacity test method shows that arabinose and melibiose are negative andmelezitose is positive.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing sodium acetate, glucose,
peptone and others, freeze-dried, and stored at 4 °C. This product, the subdivided original
strain grown in the same medium, is freeze-dried and then stored at 4 °C. The subculture of
the original strain is only one passage. This product shall not be subcultured.

618

Provisional Translation from Japanese Original

B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Enterococcus
faecalis is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is round, raised, with smoothsurface, and milky.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, cocci stained blue-violet to dark
purple are observed.
iii. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No. 9 is used as a test agar medium. The sample solution is anaerobically cultured or
aerobically at 36~38 °C for 2 days, and then the growth of the bacteria is observed
under both conditions.
v. In the test using the colony obtained by the procedure of the assay, the results of thetest on arabinose, melibiose, and melezitose using the colony obtained by theprocedure of the assay by method No. 2 of the glycolytic capacity test method showsthat arabinose and melibiose are negative and melezitose is positive.
Assay:
Preparation of sample solution: The diluent No. 1 is used as a diluent. The sample
solution is prepared at a concentration of 300~3,000 viable cell counts/mL according
to the preparation of the sample solution specified in the viable bacteria agent
quantification method.
Procedure: Medium No. 3 is used as a test agar medium. The culture medium is subjectedto the procedures of method No. 2 of viable bacteria agent quantification method, andcultured at 36~38 °C for 2 days.

619

Provisional Translation from Japanese Original(b) For manufacturing
The raw material for manufacturing of Enterococcus faecalis is cultured, and the bacterial
cells are collected, dried, and mixed with fillers. However, a mixture of Bacillus subtilis(part 4) preparation and Clostridium butyricum (part 2) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.

(146) Enterococcus faecium
Enterococcus faecium (Part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-driedEnterococcus faecium ATCC19434 strain.
Origin: The original strain is Enterococcus faecium ATCC 19434 strain which was
separated from bovine intestine in 1979.
Physical and chemical properties: This product is a facultative anaerobic Gram-positive
coccus. It breaks down lactose to produce lactate, breaks down arabinose or melibiose,but does not break down melezitose.
Confirmation test:
i. This product is applied on medium No. 3, cultured at 36~38 °C for 2 days. The colony
forming on the medium is round, raised, with smooth surface, and milky.
ii. This product is applied on medium No. 3, cultured at 36~38 °C for 2 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, cocci stained blue-violet to dark
purple are observed.
iii. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days. In the test using the colony forming on themedium by the lactic acid producing capacity test method, production of lactic acid isobserved.
iv. Medium No. 9 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured or aerobically at 36~38 °C for 2 days. The growth of thebacteria is observed under both conditions.

620

Provisional Translation from Japanese Originalv. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days. The results of the test on arabinose, melibiose,and melezitose using the colony forming on the medium by method No. 2 of theglycolytic capacity test method shows that arabinose and melibiose are positive andmelezitose is negative.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing agar, glucose, beef extract,
peptone and others, freeze-dried, and stored at 4 °C. This product is grown in a medium
containing glucose, beef extract, peptone and others, subdivided, freeze-dried, and stored at
4 °C. The subculture of the original strain is five passages or less. This product shall not be
subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofEnterococcus faecium (part 1) is cultured, and the bacterial cells are collected, added withbeta-glucan, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is round, raised, with smoothsurface, and milky.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, cocci stained blue-violet to dark
purple are observed.
iii. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No. 9 is used as a test agar medium. The sample solution is anaerobically cultured or
aerobically at 36~38 °C for 2 days, and then the growth of the bacteria is observed
under both conditions.

621

Provisional Translation from Japanese Originalv. In the test using the colony obtained by the procedure of the assay, the results of thetest on arabinose, melibiose, and melezitose using the colony obtained by theprocedure of the assay by method No. 2 of the glycolytic capacity test method showsthat arabinose and melibiose are positive and melezitose is negative.
Assay: The assay of preparation of Enterococcus faecium (part 1) is applied mutatis
mutandis.
(b) For manufacturing,
The raw material for manufacturing of Enterococcus faecium (part 1) is cultured, and thebacterial cells are collected, added with beta-glucan, dried, and mixed with fillers.
However, a mixture of Lactobacillus acidophilus (part 1) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.
Enterococcus faecium (Part 2)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-driedEnterococcus faecium 129 BIO 3B strain.
Origin: The original strain is Enterococcus faecium 129 BIO 3B strain which was separated
from yogurt in 1949.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Enterococcus faecium (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofEnterococcus faecium (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing beef, bovine liver, glucose andothers, and cryopreserved at −80 °C. For this product, the subdivided original strain grownin the same medium, is cryopreserved at −20 °C. The subculture of the original strain is two
passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofEnterococcus faecium (part 2) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.

622

Provisional Translation from Japanese OriginalConfirmation test: The confirmation test of preparation of Enterococcus faecium (part 1) isapplied mutatis mutandis
Assay: The assay of preparation of Enterococcus faecium (part 1) is applied mutatis
mutandis.
(b) For manufacturing
The raw material for manufacturing of Enterococcus faecium (part 2) is cultured, and thebacterial cells are collected, dried, and mixed with fillers. However, a mixture of
Lactobacillus acidophilus (part 6) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.
Enterococcus faecium (Part 3)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-driedEnterococcus faecium BIO-4R strain.
Origin: The original strain is Enterococcus faecium BIO-4R strain, which is Enterococcus
faecium BIO strain separated from human intestine in 1929 with the added resistance toantibacterial substances.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Enterococcus faecium (part 1).
Confirmation test:
i. This product is applied on medium No. 3, cultured at 36~38 °C for 5 days. The colony
forming on the medium is round, raised, with smooth surface, and milky.
ii. This product is applied on medium No. 3, cultured at 36~38 °C for 5 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, cocci stained blue-violet to dark
purple are observed.
iii. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 5 days. In the test using the colony forming on themedium by the lactic acid producing capacity test method, production of lactic acid isobserved.

623

Provisional Translation from Japanese Original

iv. Medium No. 9 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured or aerobically at 36~38 °C for 5 days. The growth of thebacteria is observed under both conditions.
v. Medium No. 3 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 5 days. The results of the test on arabinose, melibiose,and melezitose using the colony forming on the medium by method No. 2 of theglycolytic capacity test method shows that arabinose and melibiose are positive andmelezitose is negative.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing precipitated calcium
carbonate, lactose, peptone and others, freeze-dried, and stored at −80 °C. This product isgrown in the same medium, subdivided, and stored at 4 °C. The subculture of the original
strain is two passages or less. This product shall not be subcultured.
B. Preparation (Part 1)(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofEnterococcus faecium (part 3) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is round, raised, with smoothsurface, and milky.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, cocci stained blue-violet to dark
purple are observed.
iii. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No. 9 is used as a test agar medium. The sample solution is anaerobically cultured or624

Provisional Translation from Japanese Original

aerobically at 36~38 °C for 5 days, and then the growth of the bacteria is observed
under both conditions.
v. In the test using the colony obtained by the procedure of the assay, the results of thetest on arabinose, melibiose, and melezitose using the colony obtained by theprocedure of the assay by method No. 2 of the glycolytic capacity test method showsthat arabinose and melibiose are positive and melezitose is negative.
Assay:
Preparation of sample solution: The assay of the raw material for manufacturing ofEnterococcus faecalis is applied mutatis mutandis.
Procedure: Medium No. 3 is used as a test agar medium. The culture medium is subjectedto the procedures of method No. 2 of viable bacteria agent quantification method, andcultured at 36~38 °C for 5 days.
(b) For manufacturing,
The raw material for manufacturing of Enterococcus faecium (part 3) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
B. Preparation (Part 2)(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofEnterococcus faecium (part 3) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation (part 1) of Enterococcus faecium(part 3) is applied mutatis mutandis.
Assay: The assay of preparation (part 1) of Enterococcus faecium (part 3) is applied mutatis
mutandis.
(b) For manufacturing
The raw material for manufacturing of Enterococcus faecium (part 3) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

625

Provisional Translation from Japanese Original

Enterococcus faecium (Part 4)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-driedEnterococcus faecium FA-5 strain.
Origin: The original strain is Enterococcus faecium FA-5 strain which was separated fromhuman feces in 1975.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Enterococcus faecium (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofEnterococcus faecium (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing yeast extract, glucose peptonand others, and cryopreserved at −80 °C. For this product, the subdivided original strain
grown in the same medium, is cryopreserved at −80 °C. The subculture of the original strain
is two passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofEnterococcus faecium (part 4) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Enterococcus faecium (part 1) isapplied mutatis mutandis.
Assay: The assay of preparation of Enterococcus faecalis is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Enterococcus faecium (part 4) is cultured, and thebacterial cells are collected, dried, and mixed with fillers. However, a mixture of
Bifidobacterium thermophilum (part 2) and Lactobacillus acidophilus (part 5) preparation
shall be used.
(c) Standard of storage method
It shall be stored in a capped container.

626

Provisional Translation from Japanese Original(147) Clostridium butyricum
Clostridium butyricum (part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Clostridium butyricum MIYAIRI strain.
Origin: The original strain is Clostridium butyricum MIYAIRI strain which was separated
from human intestine in 1933.
Physical and chemical properties: This product is an obligate anaerobic Gram-positive
bacillus. It forms spores and breaks down glucose to produce butyric acid.
Confirmation test:
i. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 1~2
days. A platinumLoop amount of water is placed on a slide glass. The colony obtainedby the procedure of the assay is placed on the slide glass using a platinum wire, stirred
to be suspended, spread to the proper size, and dried at room temperature or by firefrom a distance. Then, it is passed through the flame 2~3 times to be fixed. This isused as a sample. In the test of the sample by the Gram stain, bacilli stained blue-violet
to dark purple are observed.
ii. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 2~7
days. A platinum-loop amount of water is placed on a slide glass. The colony obtainedby the procedure of the assay is placed on the slide glass using a platinum wire, stirred
to be suspended, spread to the proper size, and dried at room temperature or by firefrom a distance. Then, it is passed through the flame 2~3 times to be fixed. This isused as a sample. In the test of the sample by the spore stain method, spores stained
pale-green to green are observed.
iii. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 1~2 days. In the test using the colony
forming on the medium by the butyric acid producing capacity test method, productionof butyric acid is observed.
iv. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 1~2 days, and then the growth of the bacteria is notobserved.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing agar, beef extract, glucose, andothers, cryopreserved at −80 °C, or freeze-dried and then stored at 4 °C. This product isgrown in the same medium, subdivided, and cryopreserved at −80 °C, or freeze-dried and627

Provisional Translation from Japanese Original

then stored at 4 °C. The original strain is subcultured every five years, and this product shallnot be subcultured
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Clostridium butyricum (part 1) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.
iii. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the butyric acid producing capacity test
method, production of butyric acid is observed.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. The sample solution iscultured at 36~38 °C for 1~2 days, and then the growth of the bacteria is not observed.
Assay:
Preparation of sample solution: Approximately 1 g of this product is weighed to thedigits of 0.01 g, and the value is recorded. It is placed in a 100 mL homogenizer
vessel, added with 50 mL of diluent No. 2, and stirred at 10,000 rpm for 5 minutes.
This is used as a sample stock solution. Diluent No. 2 is hereinafter used as a diluent,and the sample solution is prepared at a concentration of 300~3,000 viable cellcounts/mL according to the preparation of the sample solution specified in the viable628

Provisional Translation from Japanese Original

bacteria agent quantification method. If necessary, the sample solution, which isheated in a water bath at 75 °C for 20 minutes and quenched in running water.
Procedure: Medium No. 1 is used as a test agar medium. The culture medium is subjectedto the procedures of method No. 2 of viable bacteria agent quantification method, andanaerobically cultured at 36~38 °C for 1~2 days.
(b) For manufacturing
The raw material for manufacturing of Clostridium butyricum (part 1) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Clostridium butyricum (part 2)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-dried Clostridium
butyricum NT strain.
Origin: The original strain is Clostridium butyricum NT strain which was separated fromhuman feces in 1955.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Clostridium butyricum (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Clostridium butyricum (part 1) is applied mutatis mutandis.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing lactose, casein peptone,
precipitated calcium carbonate and others, freeze-dried, and stored at 4 °C. This product isgrown in the same medium, subdivided, freeze-dried, and stored at 4 °C. The subculture of
the original strain is one passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Clostridium
butyricum (part 2) is cultured, and the bacterial cells are collected, dried, and mixed withfillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of the preparation of Clostridium butyricum (part1) is applied mutatis mutandis

629

Provisional Translation from Japanese Original

Assay: The assay of the preparation of Clostridium butyricum (part 1) is applied mutatis
mutandis(b) Standard of storage method
The raw material for manufacturing of Clostridium butyricum (part 2) is cultured, and thebacterial cells are collected, dried, and mixed with fillers. However, a mixture of Bacillus
subtilis (part 4) and Enterococcus faecalis preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.

(148) Bacillus coagulans
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus coagulans P-22
strain.
Origin: The original strain is Bacillus coagulans P-22 strain separated from green malt in
1949.
Physical and chemical properties: This product is a facultative anaerobic Gram-positive
bacillus. It forms spores and breaks down lactose to produce lactic acid.
Confirmation test:
i. This product is applied on medium No. 5 cultured at 36~38 °C for 2~3 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. This product is applied on medium No. 5 cultured at 36~38 °C for 3~7 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the spore stain method, spores stained pale-green
to green are observed.
iii. Medium No. 5 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2~3 days. In the test using the colony forming on the630

Provisional Translation from Japanese Original

medium by the lactic acid producing capacity test method, production of lactic acid isobserved. In addition, the culture by the lactic acid producing capacity test method is
performed for 3 days.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing agar, yeast extract, casein
peptone, and others, freeze-dried, and stored at 4 °C. This product is grown in the same
medium, subdivided, and stored at 4 °C. The subculture of the original strain is one passages
or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus
coagulans is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.
iii. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed. In addition, culturing by the lactic acidproducing capacity test method is carried out for 3 days.
Assay:
Preparation of sample solution: Approximately 1 g of this product is weighed to thedigits of 0.01 g, and the value is recorded. It is placed in a 100 mL homogenizer
vessel, added with 50 mL of diluent No. 1, and stirred at 15,000 rpm for 5 minutes.
This is used as a sample stock solution. Diluent No. 1 is hereinafter used as a diluent,631

Provisional Translation from Japanese Original

and the sample solution is prepared at a concentration of 30~300 viable cell counts/mL
according to the preparation of the sample solution specified in the viable bacteria
agent quantification method. In addition, the sample solution, which is heated in awater bath at 75 °C for 20 minutes and quenched in running water.
Procedure: Medium No. 5 is used as a test agar medium. The culture medium is subjectedto the procedures of method No. 1 of viable bacteria agent quantification method, andcultured at 36~38 °C for 2~3 days.
(b) For manufacturing,
The raw material for manufacturing of bacillus coagulans is cultured, and the bacterial
cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

(149) Bacillus subtilis
Bacillus subtilis (part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus subtilis BN strain.
Origin: The original strain is Bacillus subtilis BN strain separated from natto in 1928.
Physical and chemical properties: This product is a Gram-positive bacillus. It forms sporesand does not grow under anaerobic conditions.
Confirmation test:
i. This product is applied on medium No. 4 cultured at 36~38 °C for 1~2 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. This product is applied on medium No. 4 cultured at 36~38 °C for 3~7 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as a632

Provisional Translation from Japanese Original

sample. In the test of the sample by the spore stain method, spores stained pale-green
to green are observed.
iii. This product is applied on medium No. 4, cultured at 36~38 °C for 1~2 days. The
colony forming on the medium is collected, inoculated in a 10 mL of medium No. 7,
and anaerobically cultured at 36~38 °C for 2~3 days, and then the growth of thebacteria is not observed.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing heat treated koji, soy flour,
gelatin, and others, and cryopreserved at −80 °C. This product is grown in the same
medium, subdivided, and cryopreserved at −80 °C. The subculture of the original strain isten passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus subtilis(Part 1) is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.
iii. In the test using the the colony obtained by the procedure of the assay, inoculated into
10 ml of No. 7 medium, and cultured anaerobically at 36~38 °C for 2~3 days, and thenthe growth of the bacteria is not observed.
Assay:
Preparation of sample solution: The diluent No. 1 is used as a diluent. The sample
solution is prepared at a concentration of 30~300 viable cell counts/mL according to633

Provisional Translation from Japanese Original

the preparation of the sample solution specified in the viable bacteria agent
quantification method. If necessary, the sample solution, which is heated in a water
bath at 75 °C for 20 minutes and quenched in running water.
Procedure: Medium No. 4 is used as a test agar medium. The culture medium is subjectedto the procedures of method No. 1 of viable bacteria agent quantification method, andcultured for 1~2 days.
(b) For manufacturing,
The raw material for manufacturing of Bacillus subtilis (part 1) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Bacillus subtilis (part 2)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus subtilis C-3102
strain.
Origin: The original strain is Bacillus subtilis C-3102 strain which was separated from pig
feces in 1984.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bacillus subtilis (part 1).
Confirmation test:
i. This product is applied on medium No. 4 cultured at 36~38 °C for 1~2 days. A
platinumLoop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. This product is applied on medium No. 4 cultured at 36~38 °C for 3~7 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the spore stain method, spores stained pale-green
to green are observed.

634

Provisional Translation from Japanese Originaliii. This product is applied on medium No. 4, cultured at 36~38 °C for 1~2 days. The
colony forming on the medium is collected, inoculated in a 10 mL of medium No. 7,
and anaerobically cultured at 36~38 °C for 2~3 days, and then the growth of thebacteria is not observed.
iv. This product is applied on medium No. 6, anaerobically cultured at 36~38 °C for 1day. The grayish white, conical, and unique colony is detected 80 % or greater.
(b) Storage method and standards of subculture
The original strain is subcultured on medium No. 6 and cryopreserved at −80 °C. This
product is grown in the same medium, subdivided, and cryopreserved at −80 °C. This
product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus subtilis(part 2) is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.
iii. In the test using the the colony obtained by the procedure of the assay, inoculated into
10 ml of No. 7 medium, and cultured anaerobically at 36~38 °C for 2~3 days, and thenthe growth of the bacteria is not observed.
iv. The sample stock solution prepared by the assay is measured, and diluent No. 1 ishereinafter used as a diluent, and the sample solution is prepared at a concentration of
30~300 viable cell counts/mL according to the preparation of the sample solution
specified in the viable bacteria agent quantification method. The sample solution is635

Provisional Translation from Japanese Original

applied on medium No. 6 and cultured at 36~38 °C for 1 day. The grayish white,
conical, and specific colony is detected at 80 % or greater.
Assay:
Preparation of sample solution: The preparation of sample solution of Bacillus subtilis (part1) is applied mutatis mutandis.
Procedure: The procedure of the Bacillus subtilis (part 1) preparation is applied mutatis
mutandis. However, medium No. 4 added with glucose 1 % is used as a test agar medium.
(b) For manufacturing
The raw material for manufacturing of Bacillus subtilis (part 2) is cultured, dried, andmixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Bacillus subtilis (part 3)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus subtilis DB 9011
strain.
Origin: The original strain is Bacillus subtilis DB 9011 strain which was separated from soilin 1990.
Physical and chemical properties: The Property are the same as those of the raw material for
manufacturing of Bacillus subtilis (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of Bacillus
subtilis (part 1) is applied mutatis mutandis.
(b) Storage method and standards of subculture
The original strain is subcultured on medium No. 4 and cryopreserved at −80 °C. This
product is grown in the same medium, subdivided, and cryopreserved at −80 °C. This
product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus subtilis(part 3) is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bacillus subtilis (part 1) isapplied mutatis mutandis.

636

Provisional Translation from Japanese Original

Assay: The assay of preparation of Bacillus subtilis (part 1) is applied mutatis mutandis.
(b) For manufacturing,
The raw material for manufacturing of Bacillus subtilis (part 3) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Bacillus subtilis (part 4)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus subtilis NT strain.
Origin: The original strain is Bacillus subtilis NT strain which was separated from natto in
1985.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bacillus subtilis (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of Bacillus
subtilis (part 1) is applied mutatis mutandis.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium No. 4, freeze-dried, and stored at 4 °C.
This product is grown in the same medium, subdivided, and stored at 4 °C. The subcultureof the original strain is one passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus subtilis(part 4) is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bacillus subtilis (part 1) isapplied mutatis mutandis.
Assay: The assay of preparation of Bacillus subtilis (part 1) is applied mutatis mutandis.
(b) For manufacturing,
The raw material for manufacturing of Bacillus subtilis (part 4) is cultured, and thebacterial cells are collected, dried, and mixed with fillers.
However, a mixture of Clostridium butyricum (part 2) and Enterococcus faecalis
preparation shall be used.

637

Provisional Translation from Japanese Original(c) Standard of storage method
It shall be stored in a capped container.
Bacillus subtilis (part 5)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Bacillus subtilis JA-ZK strain.
Origin: The original strain is Bacillus subtilis JA-ZK strain which was separated from soil in
2000.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bacillus subtilis (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of Bacillus
subtilis (part 1) is applied mutatis mutandis.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing yeast extract, andcryopreserved at −70 °C or less, or freeze-dried and then stored at 2~8 °C. This product isgrown in the same medium, subdivided, and cryopreserved at −70 °C or less, or freeze-dried
and then stored at 2~8 °C. The subculture of the original strain is ten passages or less. This
product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus subtilis(part 5) is cultured, and the bacterial cells are collected, added with starch, dried, and mixed
with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bifidobacterium thermophilum(part 1) is applied mutatis mutandis
Assay: The confirmation test of preparation of Bacillus subtilis (part 1) is applied mutatis
mutandis.
(b) For manufacturing,
Preparation of sample solution: The diluent No. 1 is used as a diluent. The sample solution
is prepared at a concentration of 300~3,000 viable cell counts/mL according to the
preparation of the sample solution specified in the viable bacteria agent quantification

638

Provisional Translation from Japanese Original

method. In addition, the sample solution, which is heated in a water bath at 75 °C for 20
minutes and cooled.
Procedure: Medium No. 9 not containing the horse defibrinated blood is used as a test agar
medium. The culture medium is subjected to the procedures of method No. 2 of viablebacteria agent quantification method, and cultured at 28~30 °C for 1~2 days.
(c) Standard of storage method
It shall be stored in a capped container.

(150) Bacillus cereus
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus cereus TOYOI
strain.
Origin: The original strain is Bacillus cereus TOYOI strain which was separated from soilin 1968.
Physical and chemical properties: This product is a Gram-positive bacillus. It forms sporesand grows in the medium with chloramphenicol and polymyxin B sulfate.
Confirmation test:
i. This product is applied on medium No. 4 cultured at 36~38 °C for 1~2 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. This product is applied on medium No. 4 cultured at 36~38 °C for 3~7 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the spore stain method, spores stained pale-green
to green are observed.
iii. 100 mL of medium No. 4 and 1 mL of the antibiotic solution for viable bacteria agent
test is used as a test agar medium, cultured at 36~38 °C for 1~2 days, and then thegrowth of the bacteria is observed.

639

Provisional Translation from Japanese Original(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing agar, pepton, beef extract, andothers, freeze-dried, and stored at 4 °C. This product is grown in the same medium,
subdivided, and cryopreserved at −80 °C. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is powder in which the raw material for manufacturing of Bacillus cereus iscultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.
iii. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. Then 100 mL of medium No.
4 and 1 mL of the antibiotic solution for viable bacteria agent test is used as a test agar
medium, cultured at 36~38 °C for 1~2 days, and then the growth of the bacteria isobserved.
Assay:
Preparation of sample solution: Approximately 1 g of this product is weighed to thedigits of 0.01 g, and the value is recorded. It is placed in a 100 mL homogenizer
vessel, added with 50 mL of diluent No. 1, and stirred at 10,000 rpm for 5 minutes.
This is used as a sample stock solution. Diluent No. 1 is hereinafter used as a diluent,and the sample solution is prepared at a concentration of 30~300 viable cell counts/mL
according to the preparation of the sample solution specified in the viable bacteria

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Provisional Translation from Japanese Original

agent quantification method. If necessary the sample solution, which is heated in awater bath at 75 °C for 20 minutes and quenched in running water.
Procedure: The procedure of preparation of Bacillus subtilis is applied mutatis mutandis.
(b) For manufacturing,
The raw material for manufacturing of Bacillus cereus is cultured, and the bacterial cells
are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a lightproof airtight container.

(151) Bacillus badius
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated Bacillus badius MA001
strain.
Origin: The original strain is Bacillus badius MA 001 strain separated from the soil in
Shizuoka prefecture in 1991.
Physical and chemical properties: This product is a Gram-positive bacillus. It forms sporesand does not grow under anaerobic conditions.
Confirmation test:
i. This product is applied on the medium, in which 0.5 g (0.45~0.54 g) of yeast extract,
0.5 g (0.45~0.54 g) of sodium chloride, 0.1 g (0.05~0.14 g) of n-sodium butyrate, and
1 g (0.5~1.4 g) of agar are added with water to make 100 mL and adjusted to pH
7.7~7.9 (from (2) to (4), in B. (a). confirmation test (3) and (4), and in the procedure of
the assay, referred to as “test agar medium”), cultured at 36~38 °C for 1~2 days. A
platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. This product is applied on a test agar medium and cultured at 36~38 °C for 3~7 days.
A platinum-loop amount of water is placed on a slide glass. The colony is placed on
the slide glass using a platinum wire, stirred to be suspended, spread to the proper size,and dried at room temperature or by fire from a distance. Then, it is passed through the641

Provisional Translation from Japanese Original

flame 2~3 times to be fixed. This is used as a sample. In the test of the sample by thespore stain method, spores stained pale-green to green are observed.
iii. When this product is applied on the test agar medium, cultured at 36~38 °C for 1~2
days, the white to pale yellow-white, irregular shaped, specific colony is detected.
iv. This product is applied on the test agar medium, anaerobically cultured at 36~38 °C
for 2~3 days, and then the growth of the bacteria is not observed. This product isapplied on the medium, in which 0.5 g (0.45~0.54 g) of sodium chloride, 50 g
(49.5~50.4 g) of bovine heart extract, 1.0 g (0.95~1.04 g) of tryptose, and 1.5 g(1.45~1.54 g) of agar are added with water to make 70 mL, adjusted to pH 7.2~7.6,
and added with 30 mL of chicken egg yolk liquid (in B. (a). confirmation test (4),referred to as “egg yolk agar medium”), cultured aerobically at 36~38 °C for 1 day,and then the growth of the bacteria is not observed.
(b) Storage method and standards of subculture
The original strain is subcultured on a medium containing agar, bovine heart extract, andothers, freeze-dried, and stored at 4 °C. This product is grown in the same medium,
subdivided, and cryopreserved at −80 °C. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of Bacillus
badius is cultured, and the bacterial cells are collected, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. A platinum-loop amount of water is placed on a slide glass. The colony obtained by theprocedure of the assay is placed on the slide glass using a platinum wire, stirred to besuspended, spread to the proper size, and dried at room temperature or by fire from a
distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by Gram stain, bacilli stained blue-violet to dark
purple are observed.
ii. A platinum-loop amount of water is placed on a slide glass. The sample stock solution
prepared by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the spore stain method, sporesstained pale-green to green are observed.

642

Provisional Translation from Japanese Originaliii. The sample stock solution prepared by the assay is measured, and the diluent No. 1
added with 0.01 v/v% of polysorbate 80 is hereinafter used as a diluent to prepare thesample solution according to the preparation of sample solution by the viable bacteria
agent quantification method. This sample solution is applied on the test agar medium,cultured at 36~38 °C for 1~2 days, and then the white to pale yellow-white, irregularshaped, specific colony is detected.
iv. In the test using the colony obtained by the procedure of the assay, inoculated into thetest agar medium, and cultured anaerobically at 36~38 °C for 2~3 days, and then thegrowth of the bacteria is not observed. And in the test using the colony obtained by theprocedure of the assay, inoculated into the egg yolk agar medium, and cultured
aerobically at 36~38 °C for 1 day, and then the growth of the bacteria is not observed.
Assay:
Preparation of sample solution: Approximately 1 g of this product is weighed to thedigits of 0.01 g, and the value is recorded. It is placed in a 100 mL homogenizer
vessel, added with 50 mL of 0.1 w/v% of potassium hydroxide solution, and stirred at
10,000 rpm for 5 minutes. This is used as a sample stock solution. The diluent No. 1
added with 0.01v/v% of polysorbate 80 is used as a diluent to prepare a sample
solution at a concentration of 30~300 viable cell counts/mL according to the
preparation of sample solution by the viable bacteria agent quantification method.
Procedure: The test agar medium is used as a test agar medium. The culture medium issubjected to the procedures of method No. 1 of viable bacteria agent quantification
method, and cultured for 1~2 days.
(b) For manufacturing
The raw material for manufacturing of Bacillus badius is cultured, and the bacterial cells
are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

(152) Bifidobacterium thermophilum
Bifidobacterium thermophilum (Part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-dried
Bifidobacterium thermophilum chN-118 strain.

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Provisional Translation from Japanese Original

Origin: The original strain is Bifidobacterium thermophilum chN-118 strain which was
separated from chicken intestine in 1966.
Physical and chemical properties: This product is an obligate anaerobic Gram-positive
bacillus. It breaks down raffinose but does not break down xylose.
Confirmation test:
i. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 2~3
days and then the colony growing in the medium is milky brown to brown, a precise
circle, hemispherically raised, with a smooth surface and circumference.
ii. This product is applied on medium No. 1 and anaerobically cultured at 36~38 °C for
2~3 days. A platinum-loop amount of water is placed on a slide glass. The colony
obtained by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the Gram stain, bacilli, which arestained blue-violet to dark purple, claviform, curved, V-shaped and Y-shaped, areobserved.
iii. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2~3 days. The results of the test on xylose
and raffinose using the colony forming on the medium by method No. 1 of theglycolytic capacity test method shows that xylose are negative and raffinose ispositive.
iv. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2~3 days, and then the growth of the bacteria is notobserved.
(b) Storage method and standards of subculture
The original strain is subcultured on medium No. 1 and cryopreserved at −80 °C, orfreeze-dried and then stored at 4 °C. This product is grown in the same medium, subdivided,
freeze-dried and then stored at 4 °C. The subculture of the original strain is three passages
or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium thermophilum (part 1) is cultured, and the bacterial cells are collected,
dried, added with corn starch, shellac and lactose, granulated and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.

644

Provisional Translation from Japanese OriginalConfirmation test:
i. The colony forming by the procedure of the assay is a precise circle, hemisphericallyraised, with a smooth surface and circumference, and milky brown to brown.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli, which are stained blueviolet to dark purple, claviform, curved, V-shaped and Y-shaped, are observed.
iii. In the test using the colony obtained by the procedure of the assay, the results of thetest on xylose and raffinose using the colony forming on the medium by method No. 1of the glycolytic capacity test method shows that xylose are negative and raffinose ispositive.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. The sample solution iscultured at 36~38 °C for 2~3 days, and then the growth of the bacteria is not observed.
Assay:
Preparation of sample solution: The diluent No. 2 is used as a diluent. The sample solution
is prepared at a concentration of 300~3,000 viable cell counts/mL according to the
preparation of the sample solution specified in the viable bacteria agent quantification
methodProcedure: Medium No. 1 is used as a test agar medium. The culture medium is subjected tothe procedures of method No. 2 of viable bacteria agent quantification method, andcultured at 36~38 °C for 2~3 days.
(b) For manufacturing,
The raw material for manufacturing of Bifidobacterium thermophilum (Part 1) is cultured,and the bacterial cells are collected, dried, added with corn starch, shellac and lactose,
granulated and mixed with fillers.
However, a mixture of Lactobacillus sarivalius preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.
Bifidobacterium thermophilum (Part 2)
A. Raw material for manufacturing(a) Compositional standards

645

Provisional Translation from Japanese Original

This product is an inoculum for manufacturing proliferated and freezed Bifidobacterium
thermophilum S-501 strain.
Origin: The original strain is Bifidobacterium thermophilum S-501 strain which was
separated from pig feces in 1985.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bifidobacterium thermophilum (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofBifidobacterium thermophilum (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing ofEnterococcus faecium (Part 4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium thermophilum (part 2) is cultured, and the bacterial cells are collected,
dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bifidobacterium thermophilum(part 1) is applied mutatis mutandis
Assay: The assay of Bifidobacterium thermophilum (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Bifidobacterium thermophilum (part 2)

iscultured, and the bacterial cells are collected, dried, and mixed with fillers.
However, a mixture of Enterococcus faecium (part 4) preparation and Lactobacillus
acidophilus (part 5) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.
Bifidobacterium thermophilum (Part 3)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freezed Bifidobacterium
thermophilum SS-4 strain.
Origin: The original strain is Bifidobacterium thermophilum SS-4 strain which was
separated from pig intestine in 1966.

646

Provisional Translation from Japanese Original

Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bifidobacterium thermophilum (Part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofBifidobacterium thermophilum (Part 1) is applied mutatis mutandis.
(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing
of Bifidobacterium thermophilum (Part1) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium thermophilum (part 3) is cultured, and the bacterial cells are collected,
dried, added with corn starch, shellac and lactose, granulated and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bifidobacterium thermophilum(part 1) is applied mutatis mutandis.
Assay: The assay of Bifidobacterium thermophilum (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Bifidobacterium thermophilum (Part 3) is cultured,and the bacterial cells are collected, dried, added with corn starch, shellac and lactose,
granulated and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Bifidobacterium thermophilum (Part 4)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Bifidobacterium thermophilumWBL-4R strain.
Origin: The original strain is Bifidobacterium thermophilumWBL-4R strain which was
separated from bovine feces in 1979.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bifidobacterium thermophilum (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofBifidobacterium thermophilum (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture

647

Provisional Translation from Japanese Original

The original strain is subcultured on medium No. 1 and cryopreserved at −80 °C, orfreeze-dried and then stored at 4 °C. This product is grown in the same medium, subdivided,and cryopreserved at −80 °C, or freeze-dried and then stored at 4 °C.The subculture of theoriginal strain is three passages or less. This product shall not be subcultured.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium thermophilum (part 4) is cultured, and the bacterial cells are collected,
dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bifidobacterium thermophilum(part 1) is applied mutatis mutandis
Assay: The assay of Bifidobacterium thermophilum (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Bifidobacterium thermophilum (part 4) is cultured,and the bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

(153) Bifidobacterium pseudolongum
Bifidobacterium pseudolongum (Part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Bifidobacterium pseudolongum GSL-3 strain.
Origin: The original strain is Bifidobacterium pseudolongum GSL-3 strain which was
separated from pig feces in 1979.
Physical and chemical properties: This product is an obligate anaerobic Gram-positive
bacillus. It breaks down xylose and raffinose.
Confirmation test:
i. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 2~3
days and then the colony growing in the medium is milky brown to brown, a precise
circle, hemispherically raised, with a smooth surface and circumference.

648

Provisional Translation from Japanese Original

ii. This product is applied on medium No. 1 and anaerobically cultured at 36~38 °C for
2~3 days. A platinum-loop amount of water is placed on a slide glass. The colony
obtained by the procedure of the assay is placed on the slide glass using a platinum
wire, stirred to be suspended, spread to the proper size, and dried at room temperatureor by fire from a distance. Then, it is passed through the flame 2~3 times to be fixed.
This is used as a sample. In the test of the sample by the Gram stain, bacilli, which arestained blue-violet to dark purple, claviform, are observed.
iii. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2~3 days. The results of the test on xylose
and raffinose using the colony forming on the medium by method No. 1 of theglycolytic capacity test method shows that both are positive.
iv. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2~3 days, and then the growth of the bacteria is notobserved.
(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing
of Bifidobacterium thermophilum (Part 4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium pseudolongum (part 1) is cultured, and the bacterial cells are collected,
dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1 to 102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is a precise circle, hemisphericallyraised, with a smooth surface and circumference, and milky brown to brown.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli, which are stained blueviolet to dark purple, claviform, are observed.
iii. In the test using the colony obtained by the procedure of the assay, the results of thetest on arabinose, melibiose, and melezitose using the colony obtained by the649

Provisional Translation from Japanese Original

procedure of the assay by method No. 1 of the glycolytic capacity test method showsthat both are positive.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. The sample solution iscultured at 36~38 °C for 2~3 days, and then the growth of the bacteria is not observed.
Assay: The assay of Bifidobacterium thermophilum (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Bifidobacterium pseudolongum (part 1) iscultured, and the bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Bifidobacterium pseudolongum (Part 2)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freezed Bifidobacterium
pseudolongum M-602 strain.
Origin: The original strain is Bifidobacterium pseudolongum M-602 strain which was
separated from chicken feces in 1980.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Bifidobacterium pseudolongum (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing ofBifidobacterium pseudolongum (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing
of Enterococcus faecium (Part 4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing ofBifidobacterium pseudolongum (part 2) is cultured, and the bacterial cells are collected,
dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Bifidobacterium pseudolongum(part 1) is applied mutatis mutandis
Assay: The assay of Bifidobacterium thermophilum (part 1) is applied mutatis mutandis.

650

Provisional Translation from Japanese Original(b) For manufacturing
The raw material for manufacturing of Bifidobacterium pseudolongum (part 2) iscultured, and the bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

(154) Lactobacillus acidophilus
Lactobacillus acidophilus (Part 1)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-dried
Lactobacillus acidophilus ATCC 33199 strain.
Origin: The original strain is Lactobacillus acidophilus ATCC 33199 strain separated fromthe avian crop (chicken) in 1980.
Physical and chemical properties: This product is a facultative anaerobic Gram-positive
bacillus. It breaks down lactose to produce lactic acid and breaks down amygdalin andcellobiose.
Confirmation test:
i. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 2days and then the colony growing in the medium is milky brown to brown, slightly
irregular or precise circle, hemispherically or slightly raised, with a smooth or roughsurface and circumference.
ii. This product is applied on medium No. 1 and anaerobically cultured at 36~38 °C for 2days. A platinum-loop amount of water is placed on a slide glass. The colony obtainedby the procedure of the assay is placed on the slide glass using a platinum wire, stirred
to be suspended, spread to the proper size, and dried at room temperature or by firefrom a distance. Then, it is passed through the flame 2~3 times to be fixed. This isused as a sample. In the test of the sample by the Gram stain, bacilli or long bacilli,
which are stained blue-violet to dark purple are observed.
iii. Medium No. 9 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days, and then the growth of the bacteria is poor.
iv. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days, and then the growth of the bacteria is observed.
v. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2 days. In the test using the colony forming

651

Provisional Translation from Japanese Originalon the medium by the lactic acid producing capacity test method, production of lactic
acid is observed.
vi. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2 days. The results of the test using thecolony growing in the medium on amygdalin and cellobiose by method No. 1 of theglycolytic capacity test method shows that both are positive.
(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing
of Enterococcus faecium (Part 1) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is cultured, and the bacterial cells are collected, addedwith beta-glucan, dried, and mixed with fillers.
Content: When this product is determined, it contains 10−1 to 102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is milky brown to brown, slightly
irregular or precise circle, hemispherically or slightly raised, with a smooth or roughsurface and circumference.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli or long bacilli, which arestained blue-violet to dark purple, are observed.
iii. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No. 9 is used as a test agar medium. The sample solution is cultured at 36~38 °C for 2days, and then the growth of the bacteria is poor.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No.1 is used as a test agar medium. The sample solution is cultured at 36~38 °C for 2days, and then the growth of the bacteria is observed.

652

Provisional Translation from Japanese Originalv. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed
vi. In the test using the colony obtained by the procedure of the assay, the results of thetest using the colony growing in the medium on amygdalin and cellobiose by method
No. 1 of the glycolytic capacity test method shows that both are positive.
Assay:
Preparation of sample solution: The preparation of sample solution of Bifidobacterium
thermophilum (part 1) is applied mutatis mutandis.
Procedure: Procedure: Medium No. 1 is used as a test agar medium. The culture medium
is subjected to the procedures of method No. 2 of viable bacteria agent quantification
method, and cultured at 36~38 °C for 2days.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus acidophilus (part 1) is cultured, andthe bacterial cells are collected, added with beta-glucan, dried, and mixed with fillers.
However, a mixture of Enterococcus faecium (part 1) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.
Lactobacillus acidophilus (Part 2)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Lactobacillus acidophilus GAL-2 strain.
Origin: The original strain is Lactobacillus acidophilus GAL-2strain which was separated
from chicken feces in 1979.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Lactobacillus acidophilus (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of Bifidobacterium thermophilum (part4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards653

Provisional Translation from Japanese Original

This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 2) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10|−1 to 102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Lactobacillus acidophilus (part 1)
is applied mutatis mutandis
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus acidophilus (part 2) is cultured, andthe bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Lactobacillus acidophilus (Part 3)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Lactobacillus acidophilus GBL-2 strain.
Origin: The original strain is Lactobacillus acidophilus GBL-2 strain which was separated
from bovine feces in 1979.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Lactobacillus acidophilus (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of Bifidobacterium thermophilum (part4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 3) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.

654

Provisional Translation from Japanese OriginalConfirmation test: The confirmation test of preparation of Lactobacillus acidophilus (part 1)
is applied mutatis mutandis
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus acidophilus (part 3) is cultured, andthe bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Lactobacillus acidophilus (Part 4)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze or freeze-dried
Lactobacillus acidophilus GSL-2 strain.
Origin: The original strain is Lactobacillus acidophilus GSL-2 strain which was separated
from pig feces in 1979.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Lactobacillus acidophilus (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of Bifidobacterium thermophilum (part4) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 4) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Lactobacillus acidophilus (part 1)
is applied mutatis mutandis
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus acidophilus (part 4) is cultured, andthe bacterial cells are collected, dried, and mixed with fillers.

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Provisional Translation from Japanese Original(c) Standard of storage method
It shall be stored in a capped container.
Lactobacillus acidophilus (Part 5)
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freezed Lactobacillus
acidophilus LAC-300 strain.
Origin: The original strain is Lactobacillus acidophilus LAC-300 strain which was
separated from human feces in 1977.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Lactobacillus acidophilus (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of Enterococcus faecium (part 4) isapplied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 5) is cultured, and the bacterial cells are collected, dried, andmixed with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Lactobacillus acidophilus (part 1)
is applied mutatis mutandis
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus acidophilus (part 5) is cultured, andthe bacterial cells are collected, dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.
Lactobacillus acidophilus (Part 6)
A. Raw material for manufacturing(a) Compositional standards

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Provisional Translation from Japanese Original

This product is an inoculum for manufacturing proliferated and freezed Lactobacillus
acidophilus M-13 strain.
Origin: The original strain is Lactobacillus acidophilus M-13 strain which was separated
from pig feces in 1970.
Physical and chemical properties: The physical and chemical properties are the same as
those of the raw material for manufacturing of Lactobacillus acidophilus (part 1).
Confirmation test: The confirmation test of the raw material for manufacturing of
Lactobacillus acidophilus (part 1) is applied mutatis mutandis(b) Storage method and standards of subculture
The storage method and standards of subculture of Enterococcus faecium (part 2) isapplied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus acidophilus (part 6) is cultured and the bacterial cells are collected, addedwith L-arginine hydrochloride, dried, and added with fillers.
Content: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test: The confirmation test of preparation of Lactobacillus acidophilus (part 1)
is applied mutatis mutandis
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
For manufacturing, the raw material for manufacturing of Lactobacillus acidophilus (part6) is cultured and the bacterial cells are collected, added with L-arginine hydrochloride,
dried, and mixed with fillers.
(c) Standard of storage method
It shall be stored in a capped container.

(155) Lactobacillus salivarius
A. Raw material for manufacturing(a) Compositional standards
This product is an inoculum for manufacturing proliferated and freeze-dried
Lactobacillus salivarius chN-426 strain.
Origin: The original strain is Lactobacillus salivarius chN-426 strain which was separated
from chicken intestine in 1966.

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Provisional Translation from Japanese Original

Physical and chemical properties: This product is a facultative anaerobic Gram-positive
bacillus. It breaks down lactose to produce lactic acid and not breaks down amygdalinand cellobiose.
Confirmation test:
i. This product is applied on medium No. 1, anaerobically cultured at 36~38 °C for 2days and then the colony growing in the medium is milky brown to brown, slightly
irregular or precise circle, hemispherically or slightly raised, with a smooth or roughsurface and circumference.
ii. This product is applied on medium No. 1 and anaerobically cultured at 36~38 °C for 2days. A platinum-loop amount of water is placed on a slide glass. The colony obtainedby the procedure of the assay is placed on the slide glass using a platinum wire, stirred
to be suspended, spread to the proper size, and dried at room temperature or by firefrom a distance. Then, it is passed through the flame 2~3 times to be fixed. This isused as a sample. In the test of the sample by the Gram stain, bacilli or long bacilli,
which are stained blue-violet to dark purple, are observed.
iii. Medium No. 9 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days, and then the growth of the bacteria is poor.
iv. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and cultured at 36~38 °C for 2 days, and then the growth of the bacteria is observed.
v. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2 days. In the test using the colony formingon the medium by the lactic acid producing capacity test method, production of lactic
acid is observed.
vi. Medium No. 1 is used as a test agar medium. This product is applied on the medium
and anaerobically cultured at 36~38 °C for 2 days. The results of the test using thecolony growing in the medium on amygdalin and cellobiose by method No. 1 of theglycolytic capacity test method shows that both are negative.
(b) Storage method and standards of subculture
The storage method and standards of subculture of the raw material for manufacturing
of Bifidobacterium thermophilum (Part 1) is applied mutatis mutandis.
B. Preparation(a) Compositional standards
This product is pieces or powder in which the raw material for manufacturing of
Lactobacillus salivarius is cultured, and the bacterial cells are collected, dried, added withcorn starch, shellac and lactose, granulated and mixed with fillers.

658

Provisional Translation from Japanese OriginalContent: When this product is determined, it contains 10−1~102 times/g as much as theviable cell count on the label.
Confirmation test:
i. The colony forming by the procedure of the assay is milky brown to brown, slightly
irregular or precise circle, hemispherically or slightly raised, with a smooth or roughsurface and circumference.
ii. A platinum-loop amount of water is placed on a slide glass. The colony obtained by
the procedure of the assay is placed on the slide glass using a platinum wire, stirred tobe suspended, spread to the proper size, and dried at room temperature or by fire froma distance. Then, it is passed through the flame 2~3 times to be fixed. This is used as asample. In the test of the sample by the Gram stain, bacilli or long bacilli, which arestained blue-violet to dark purple, claviform, are observed.
iii. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No. 9 is used as a test agar medium. The sample solution is cultured at 36~38 °C for 2days, and then the growth of the bacteria is poor.
iv. The sample solution prepared by the assay is subjected to the procedure of method
No. 2 of the viable bacteria agent quantification method. In this procedure, medium
No.1 is used as a test agar medium. The sample solution is cultured at 36~38 °C for 2days, and then the growth of the bacteria is observed.
v. In the test using the sample stock solution 0.1 mL prepared by the assay or the colony
obtained by the procedure of the assay by the lactic acid producing capacity test
method, production of lactic acid is observed
vi. In the test using the colony obtained by the procedure of the assay, the results of thetest using the colony growing in the medium on amygdalin and cellobiose by method
No. 1 of the glycolytic capacity test method shows that both are negative.
Assay: The assay of Lactobacillus acidophilus (part 1) is applied mutatis mutandis.
(b) For manufacturing
The raw material for manufacturing of Lactobacillus salivarius is cultured, and thebacterial cells are collected, dried, added with corn starch, shellac and lactose, granulated
and mixed with fillers.
However, a mixture of Bifidobacterium thermophilum (part 1) preparation shall be used.
(c) Standard of storage method
It shall be stored in a capped container.

659

Provisional Translation from Japanese Original(156) Benzoic acid
A.Active ingredients for production(a)Compositional standards
Content: Benzoic acid, when dried, contains not less than 99.5% of benzoic acid (C7H6O2).
Physical and chemical properties
i. Benzoic acid occurs as white crystals or crystalline powder.
ii. Melting point: 121 ~ 123°C
Identification: Dissolve 1.0 g (0.95 ~ 1.04 g) of benzoic acid in 8 mL of 1 mol/L sodiumhydroxide TS, add water to make 100 mL, and add ferric chloride TS. A light yellow-red
precipitate is formed, which changes to a white precipitate upon the addition of dilutehydrochloric acid.
Purityi. Lead: Weigh 2.0 g (1.95 ~ 2.04 g) of benzoic acid, place it in a platinum, quartz, or
porcelain crucible, and heat gradually. Before carbonization begins, stop the heating, add 1
mL of sulfuric acid, and gradually raise the temperature until the sample is carbonized togenerate no white fumes of sulfuric acid.If necessary, add more sulfuric acid and heat it
until most of the sample is carbonized. Put the lid on the container loosely, heat it
gradually, and ignite to incinerate at 450 ~ 600 °C. If carbides remain, crush with a glassrod where necessary, moisten with 1 mL of sulfuric acid (1 → 4) and 1 mL of nitric acid,heat until no white fumes are evolved, and ignite to incinerate completely. To the residue,
add 10 mL of hydrochloric acid (1 → 4), heat on a water bath, and evaporate to dryness.
To the residue, add a small amount of nitric acid (1 → 100), and warm to dissolve it. Aftercooling, add nitric acid (1 → 100) to make exactly 10 mL, and use this solution as the
sample solution. Measure 0.4 mL of standard lead solution using a volumetric pipette,
transfer to a 10-mL volumetric flask, add nitric acid (1 → 100) to the marked line to make
10 mL, and use this solution as the standard solution. Perform the test for lead with thesample solution and standard solution as directed under Lead Test (Method 1 of Atomic
Absorption Spectrophotometry): the amount of lead shall be not more than 2μg/g.
ii. Arsenic: Prepare the sample solution with 0.5 g (0.45 ~ 0.54 g) of benzoic acid according
to Method 3 of the Arsenic Test, and perform the test for arsenic by the method using
Apparatus A: the color of the absorbing liquid shall have no more color than the standard
color (not more than 3 μg/g).
iii. Phthalic acid: To 0.10 g (0.095 ~ 0.104 g) of benzoic acid, add 1 mL of water and 1 mLof resorcinol-sulfuric acid TS, heat in an oil bath at 120 ~125 °C to evaporate water, andheat for another 90 minutes. After cooling, dissolve the residue in 5 mL of water, add 10
mL of a sodium hydroxide solution (43 → 500) to 1 mL of this solution, shake, and use

660

Provisional Translation from Japanese Original

this solution as the sample solution.Separately, dissolve 61 mg of potassium hydrogen
phthalate (standard reagent) in water to make exactly 1000 mL. Pipet 1 mL of this solution
exactly, add 1 mL of resorcinol-sulfuric acid TS, proceed in the same manner as for the
sample solution, and use this solution as the control solution. Examine the sample solution
and the control solution under 470 ~ 490 nm light: the green fluorescence of the sample
solution shall have no more color than the control solution (not more than 500 μg/g).
iv. Related substances: Weigh 5.0 g (4.95 ~5.04 g) of benzoic acid, transfer it into a 25-mL
volumetric flask, dissolve it with 1 mL of propyl benzoate-dimethylformamide TS, add
dimethylformamide to make 25 mL, and use this solution as the sample solution.
Separately, weigh 40 mg each of propyl benzoate, biphenyl, 2-methylbiphenyl, 3methylbiphenyl, 4-methylbiphenyl, and benzyl benzoate, and 20 g of benzoic acid, transfer
them into a 100-mL volumetric flask, add about 50 mL of dimethylformamide to dissolve
them, add dimethylformamide to make 100 mL, and use this solution as the standard
solution. Perform the test with 1μL each of the sample solution and standard solution as
directed under Gas Chromatography according to the following conditions. Based on thechromatogram obtained, identify the peaks of biphenyl, 2-methylbiphenyl, 3methylbiphenyl, 4-methylbiphenyl, and benzyl benzoate appearing on the chromatogramof the sample solution by using the chromatogram of the standard solution, and determine
the peak area Ac1. Treat the unidentified peak that appears between the peaks of 2methylbiphenyl and benzoic acid as the peak of the dimethylbiphenyl isomer, anddetermine the content Ac1 of the peak area. Based on these values and the peak area Ai1of propyl benzoate determined from the chromatogram of the sample solution, calculate
the content of the related substances of benzoic acid (biphenyl, 2-methylbiphenyl, 3methylbiphenyl, 4-methylbiphenyl, benzyl benzoate, and dimethylbiphenyl isomer) in thesample using the following equation: the total content shall not exceed 100 μg/g.
Ai1: Peak area of propyl benzoate in the sample solution
Ac1: Peak area of each related substance in the sample solution
Ms1: Amount (g) of sample in the sample solution
Mi1: Amount (mg) of propyl benzoate in the sample solution
RFc: Response factor of each related substance
The response factor (RFc) of each related substance shall be determined using thefollowing equation:
As for the RFc of the dimethylbiphenyl isomer, use the RFc of 3-methylbiphenyl.
Ai2: Peak area of propyl benzoate in the standard solution
Ac2: Peak area of each related substance in the standard solution
Mc2: Amount (mg) of each related substance in the standard solution

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Provisional Translation from Japanese Original

Mi2: Amount (mg) of propyl benzoate in the standard solution
Operating conditions
Detector: Hydrogen flame ionization detector
Column: A fused silica column 0.32 mm in inside diameter and 15 to 30 m in length,coated with polyethylene glycol modified with nitroterephthalic acid to a thickness of 1
μm.
Column temperature: Hold the temperature at 100 °C for one minute, then raise it to
210 °C at a rate of 3 °C per minute.
Injection port temperature: 240 °C (if a temperature increase program can be set, raise thetemperature to 270°C at a rate of 12°C per second with an initial temperature of 80 °C)
Detector temperature: 240 ~ 250 °C
Carrier gas: Helium
Flow rate: About 3 mL per minute (If pressure control is possible, hold the pressure at 47
kPa for 4.35 minutes and increase it to 58 kPa at a rate of 0.28 kPa per minute.)
Injection method: Split
Split ratio: 1:10
Loss on drying: Not more than 0.5% (1 g, silica gel, 3 hours)
Residue on ignition: Not more than 0.05% (1 g)Assay: Weigh about 0.5 g of benzoic acid, previously dried, to the order of 0.001 g, record
the value, add 25 mL of neutralized ethanol and 25 mL of water to dissolve, and titrate with a
0.1 mol/L sodium hydroxide solution (indicator: 3 drops of phenolphthalein TS).
Each mL of 0.1 mol/L sodium hydroxide solution = 12.21 mg C7H6O2(b)Standards for manufacturing method
Manufacture by air oxidation of toluene.
(c)Standards for preservation method
Store in an airtight container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of benzoic acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of benzoic acid isapplied mutatis mutandis.

662

Provisional Translation from Japanese Original(157) Calcium formate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following drying, it contains 98.0 % or over ofcalcium formate (Ca(HCOO)2).
Physical and chemical properties:
i. This product is white to pale yellow crystals or powder.
ii. This product is easy to dissolve in water and hardly dissolves in methanol.
Confirmation test:
i. The solution of this product in water (1 → 10) gives the qualitative reactions i. to iii. of
calcium salt.
ii. 1 mL of the solution, in which approximately 0.5 g of this product is dissolved in 5 mLof water, is measured, added with 0.5 mL of hydrochloric acid, and added with 20 mg(19.5~20.4 mg) of magnesium powder in several times. When bubbles disappear, it isadded with 3 mL of sulfuric acid (3 → 5) and 10 mg (9.5~10.4 mg) of chromotropic
acid and vigorously shaken. It is heated in a water bath for approximately 10 minutes,
and then it is red to purple.
Purity test:
i. Clarity and color of solution: 1.0 g (0.95~1.04 g) of this product is dissolved in 100 mLof water. The resulting solution shall be colorless to pale yellow and transparent.
ii. pH: The pH of solution in water (1→100) of this product shall be 7.0~8.5.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (20 mg/kg or
less).
iv. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 1 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (4 mg/kg or less).
Water content: 0.2 % or less (Water metering method).
Assay: Approximately 2.0 g of this product is weighed to the digits of 0.001 g, and the value
is recorded. It is dissolved with water 50 mL and titrated with 1 mol/L hydrochloric acid(potentiometric titration). A blank test is performed by the same method and corrections
are made.

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Provisional Translation from Japanese Original

1 mL of 1 mol/L hydrochloric acid = 65.01 mg Ca(HCOO)2(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of calcium formate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of calcium formate
is applied mutatis mutandis.

(158) Sodium gluconate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following drying, it contains 98.0~102.0 % of
sodium gluconate (C6H11O7Na).
Physical and chemical properties: This product is white to yellowish-white crystalline
powder or particles.
Confirmation test:
i. The solution of this product in water (1 → 20) gives the qualitative reaction i. and iii.
of sodium salt.
ii. When an infrared absorption spectrum of this product is measured by the paste methodof the infrared absorption spectroscopy, the absorption is observed at near 3,520~3,560
cm−1, 3,410~3,450 cm−1, 3,290~3,330 cm−1, 1,580~1,660 cm−1, and 1,080~1,100 cm−1.
Purity test:
i. Clarity and color of solution: This product 1.0 g (0.95~1.04 g) is dissolved in 10 mL ofwater. The resulting solution shall be colorless and almost clear.
ii. pH: The pH the solution of this product in water (1 → 10) shall be 6.2~7.8.
iii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (20 mg/kg or
less).

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Provisional Translation from Japanese Original

iv. Arsenic: 0.5 g (0.45~0.54 g) of this product is weighed, and a sample solution isprepared by Method No. 1 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (4 mg/kg or less).
Loss on drying: 0.30 % or less (2 g, 105 °C, 2 hours)
Assay: This product is dried and appropriately 0.15 g of it is weighed to the digits of 0.001
g, and the values is recorded. It is added with 75 mL of acetic acid and titrated with 0.1
mol/L perchloric acid test solution (indicator: 10 drops of quinaldine red). In this case, theend point of titration is the time when the red color of the solution disappears. A blanktest is performed by the same method and corrections are made.
1 mL of 0.1 mol/L perchloric acid = 21.81 mg C6H11O7Na(b) Standard of storage method
It shall be stored in a capped container.
B. Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of sodium gluconate
are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of sodium
gluconate is applied mutatis mutandis.

(158) Potassium diformate
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined following drying, it contains 95.0 % or over ofpotassium diformate (C2H3O4K).
Physical and chemical properties: This product is white crystalline powder with no odor or aslight specific odor.
Confirmation test:
i. The pH of the solution of this product in water (1 → 100) shall be 3.3~4.3.
ii. 1 g (0.95~1.04 g) of this product is added with 10 mL of water and filtered. Thissolution 1 mL is added with 1 mL of ethanol and 3 drops of sulfuric acid, heated in awater bath, and then the odor of ethyl formate occurs.
iii. 1 g (0.95~1.04 g) of this product is added with 10 mL of water and filtered. Thissolution is added with lead acetate solution and then white crystalline precipitation is665

Provisional Translation from Japanese Original

generated. In addition, when it is added with silver nitrate test solution and heated, it
becomes cloudy within 30 seconds.
iv. This product gives the qualitative reaction i. of potassium salt.
Purity test:
i. Melting point: The melting point of this product shall be 108~109 °C.
ii. Heavy metal: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 2 of the heavy metals test method. A control solution isprepared using 2.0 mL of lead standard solution by the method. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (20 mg/kg or
less).
iii. Arsenic: 1.0 g (0.95~1.04 g) of this product is weighed, and a sample solution isprepared by Method No. 3 of the arsenic test method. When the solution is tested by
the arsenic test method using device A, the color of absorbing solution shall not be
darker than the standard color (2 mg/kg or less).
Water content: 1.0 % or less (Direct titration method).
Assay: Approximately 2 g of this product is weighed to the digits of 0.01 g and the value isrecorded. It is dissolved with water, transferred to a 500 mL volumetric flask, added withwater to the graduation line to make 500 mL. 20 mL of this solution measured using a
volumetric pipette is transferred to a stoppered Erlenmeyer flask, and while shaking
added with 18~19 mL of sodium carbonate test solution and 50 mL of 0.02 mol/L
potassium permanganate using a volumetric pipette. It is heated at 80 °C for 5 minuteswith occasionally shaking, quenched, while shaking added with 1 g (0.5~1.4 g) ofpotassium iodide and 15 mL of 2 mol/L sulfuric acid test solution, and titrated with 0.1
mol/L sodium thiosulfate solution (indicator: starch test solution 1 mL). A blank test is
performed by the same method and corrections are made.
Amount of potassium diformate (mg) = Titer of 0.1 mol/L sodium thiosulfate solution(mL) × 3.254(b) Standard of manufacturing method
For manufacturing, potassium diformate crystals produced by reaction of formic acid andpotassium formate are added with vegetable oil, silica gel and others.
(c) Standard of storage method
It shall be stored in an air tight container.
B. Preparation(a) Compositional standards

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Provisional Translation from Japanese Original

The compositional standards of the raw material for manufacturing of potassium
diformate are applied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of potassium
diformate is applied mutatis mutandis.

(160) Fumaric acid
A. Raw material for manufacturing(a) Compositional standards
Content: When this product is determined, it contains 99.0 % or over of fumaric acid
(C4H4O4).
Physical and chemical properties: This product is white crystalline powder without odor,
and with a specific sourness.
Confirmation test:
i. This product sublimes by heating.
ii. When this product is dried at 105 °C for 3 hours and its melting point is measured, thatis 287~302 °C (degradation).
iii. When 0.5 g (0.45~0.54 g) of this product is added with 10 mL of water, dissolved byboiling, and then added with 2~3 drops of thermal bromine test solution, the color of
the solution disappears.
iv. 50 mg (49.5~50.4 mg) of this product is placed in a test tube, added with 2~3 mg ofresorcin and 1 mL of sulfuric acid, shaken, heated at 120~130 °C for 5 minutes,
allowed to cool, and added with water to make 5 mL. While cooling, this solution isadded with drops of sodium hydroxide solution (2 → 7) to make it alkaline, and addedwith water to make 10 mL. The resulting solution emits green-blue fluorescence underultraviolet light.
Purity test:
i. Clarity and color of solution: 0.5 g (0.45~0.54 g) of this product is weighed anddissolved with 10 mL of sodium hydroxide solution (1 → 25). The resulting solution
shall be colorless and clear.
ii. Sulfate: 1.0 g (0.95~1.04 g) of this product is weighed, added with 30 mL of water,
shaken, added with 1 drop of phenolphthalein test solution, and added with drops ofammonia test solution until the solution exhibits slightly red. This is used as a sample
solution. A control solution is prepared using 0.2 mL of 0.005 mol/L sulfuric acid.
When the sample solution and the control solution are tested by the qualitative reaction

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Provisional Translation from Japanese Original

test method of sulfate, the opacity of the sample solution is shall not be darker thanthat of the control solution (0.01 % or less).
iii. Heavy metal: 2.0 g (1.95~2.04 g) of this product is weighed, added with 30 mL ofwater, shaken, added with 1 drop of phenolphthalein test solution, added with drops ofammonia test solution until the solution exhibits slightly red, and added with 2 mL ofacetic acid (1 → 20) and water to make 50 mL. This is used as a sample solution. A
control solution is prepared using 2.0 mL of lead standard solution. When the sample
solution and the control solution are tested by the heavy metals test method, the colorof the sample solution shall not be darker than that of the control solution (10 mg/kg or
less).
iv. Arsenic: 0.5 g (0.45~0.54 g) of the product is weighed, added with 10 mL of water,
dissolved by heating, and allowed to cool. This is used as a sample solution. When thesample solution is tested by the arsenic test method using device A, except, 10 mL ofacidic stannous chloride test solution and 3 g (2.5~3.4 g) of arsenic-free zinc are used,the color of the absorbing solution shall not darker than that of standard, (4 mg/kg or
less).
Ignition residue: 0.05 % or less (5 g)Assay: Approximately 1 g of this product is weighed to the digits of 0.01 g and the value isrecorded. It is dissolved with water, transferred to a 250 mL volumetric flask, and addedwith water to the graduation line to make 250 mL. This solution 25 mL is measured using
a volumetric pipette and titrated with 0.1 mol/L sodium hydroxide solution (indicator: 2drops of phenolphthalein test solution).
0.1 mol/L sodium hydroxide solution = 5.804 mg of C4H4O4(b) Standard of storage method
It shall be stored in a capped container.
B.Preparation(a) Compositional standards
The compositional standards of the raw material for manufacturing of fumaric acid areapplied mutatis mutandis.
(b) Standard of storage method
The standard of storage method of the raw material for manufacturing of fumaric acid isapplied mutatis mutandis.

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Provisional Translation from Japanese Original(161) Formic Acid/Propionic Acid
Preparation(a) Compositional standards
This product is liquid, in which the raw material for manufacturing of formic acid or its
preparation and the raw material for manufacturing of propionic acid or its preparation are
mixed and if necessary added with ammonia (limited to that meeting the standards of foodadditives), caprylic acid or food dye.
Content: This product is determined to contain formic acid (CH2O2) corresponding to
97~103 % of the amount on the label and propionic acid (C3H6O2) corresponding to
95~105 % of the amount on the label.
Confirmation test:
i. The solution of this product in water (1 → 10) is acidic.
ii. This product 1 mL is added with 1 mL of ethanol and 3 drops of sulfuric acid, heatedin a water bath, and then the odor of carboxylate occurs.
iii. This product is added with lead acetate test solution and then while crystalline
precipitation occurs. In addition, when it is added with silver nitrate test solution andheated, it becomes cloudy within 30 seconds.
Assay:
i. Formic acid: The amount of this product containing approximately 100 mg of formic
acid (CH2O2) is weighed to three significant digits and the value is recorded. It isdissolved with water, transferred to a 100 mL volumetric flask, added with water to thegraduation line to make 100 mL. This is used as a sample solution. Separately,
approximately 150 mg of sodium formate for assay is measured to the digits of 1 mg
and the value is recorded. It is dissolved with water, transferred to a 100 mL
volumetric flask, added with water to the graduation line to make 100 mL. This is used
as a standard solution. 20 µL each of the sample and standard solution are tested by theliquid chromatography under the following conditions. The peak areas are measured
from the obtained chromatograms.
Amount of formic acid (CH2 O2 ) (mg)= Collected amount of sodium formate for assay (mg) ×AT
1,478 × AS

AT: Peak area of the sample solution

AS: Peak area of the standard solution
Operating condition
Detector: Ultraviolet absorptiometer (measurement wavelength: 210 nm)

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Provisional Translation from Japanese Original

Column: A stainless tube (inner diameter: 8 mm, length: 300 mm) is filled with 8~11
µm of strong-acid cation exchange resin.
Column temperature: 50 °C
Mobile phase: Perchloric acid 0.85 mL is added with 1,000 mL of water.
Flow rate: Approx. 1.0 mL/min.
Column selection: A column is used when: 150 mg (149.5~150.4 mg) of sodium
formate for assay and 32 mg (31.5~32.4 mg) of sodium propionate for assay areweighed, dissolved with water, transferred to a 100 mL volumetric flask, and addedwith water to the graduation line to make 100 mL. When 20 µL of this solution is
subjected to the procedure under the above conditions, formic acid and propionicacid are eluted in this order, and the separation degree is 2.0 or greater.
ii. Propionic acid: The amount of this product containing approximately 25 mg of propionicacid (C3H6O2) is weighed to three significant digits and the value is recorded. It isdissolved with water, transferred to a 100 mL volumetric flask, and added with water tothe graduation line to make 100 mL. This is used as a sample solution. Separately,
approximately 32 mg of sodium propionate for assay is weighed to the digits of 0.01 mg
and the value is recorded. It is dissolved with water, transferred to a 100 mL volumetricflask, and added with water to the graduation line to make 100 mL. This is used as astandard solution. 20 µL each of the sample and standard solutions are tested by theliquid chromatography under the conditions of i. The peak areas are measured from theobtained chromatograms.
Amount of formic acid (C3 H6O2 ) (mg)= Collected amount of sodium formate for assay (mg) ×AT
1,297 × AS

AT: Peak area of the sample solution

AS: Peak area of the standard solution(b) Standard of storage method
It shall be stored in an acid resistant airtight capped container.
(c) Standards of the label
The following words shall be written on the immediate container or the immediatewrapper of this product.
“使用上の注意 この飼料添加物は、pH が低いことから取扱いに注意すること。”
Precaution: This feed additive requires careful handling due to its low pH.

670

Provisional Translation from Japanese Original(162) Vitamin AD
A. Preparation (Part 1: liquid)(a) Compositional standards
This is oil liquid or water-soluble liquid, which is a mixture of the raw material for
manufacturing or preparations of vitamin A oil or vitamin A powder and the raw material
for manufacturing or preparations of cholecalciferol, ergocalciferol, vitamin D3 oil orvitamin D powder in hydrogenated oils, higher saturated fatty acids, fatty acids, vegetable
oil or animal fats.
Content: The assay shows that this product contains vitamin A (retinol) corresponding to
90~130 % of the unit on the label and cholecalciferol (C27H44O) or ergocalciferol(C28H44O) corresponding to 90~130 % of the unit on the label.
Confirmation test:
i. Vitamin A: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
ii. Cholecalciferol or ergocalciferol: The confirmation test of the raw material for
manufacturing of vitamin D powder is applied mutatis mutandis.
Assay:
i. Vitamin A: It tested by Method No. 2 of the vitamin A assay.
ii. Cholecalciferol or ergocalciferol: It tested by method of the vitamin D assay.
(b) Standard of storage method
It shall be stored in a lightproof airtight container making it almost full or with nitrogen
replaced with air.
B. Preparation (Part 2: powder)(a) Compositional standards
This is powder or particles, which is a mixture of the raw material for manufacturing or
preparations of vitamin A oil or vitamin A powder and the raw material for manufacturing
or preparations of cholecalciferol, ergocalciferol, vitamin D3 oil or vitamin D powder infillers.
Content: The assay shows that this product contains vitamin A (retinol) corresponding to
90~130 % of the unit on the label and cholecalciferol (C27H44O) or ergocalciferol(C28H44O) corresponding to 90~130 % of the unit on the label.
Confirmation test:
i. Vitamin A: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
ii. Cholecalciferol or ergocalciferol: The confirmation test of the raw material for
manufacturing of vitamin D powder is applied mutatis mutandis.

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Provisional Translation from Japanese Original

Assay:
i. Vitamin A: The amount of this product corresponding to 500 units or more of Vitamin
A and containing 1 or less of fat and oil is weighed to three significant digits and thevalue is recorded. It is placed in a flask, added with 2 mL of water, heated for a whilewith shaking, and added with 30 mL of aldehyde-free ethanol and 1 mL of ethanol
solution of pyrogallol (1 → 10), and from here the test by Method No. 2 of the vitamin
A assay is performed.
ii. Cholecalciferol or ergocalciferol: It tested by method of the vitamin D assay.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

(163) Vitamin ADE
A. Preparation (Part 1: liquid)(a) Compositional standards
This is oil liquid or water-soluble liquid, which is a mixture of the raw material for
manufacturing or preparations of vitamin A oil or vitamin A powder, the raw material for
manufacturing or preparations of cholecalciferol, ergocalciferol, vitamin D3 oil or vitamin D
powder, and the raw material for manufacturing or preparations of dl-α-tocopherol acetate
or vitamin E powder in hydrogenated oils, higher saturated fatty acids, fatty acids, vegetable
oil or animal fats.
Content: The assay shows that this product contains vitamin A (retinol) corresponding to
90~130 % of the unit on the label, cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
corresponding to 90~130 % of the unit on the label, and dl-α-tocopherol acetate(C31H52O3) corresponding to 90~120 % of the unit on the label.
Confirmation test:
i. Vitamin A: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
ii. Cholecalciferol or ergocalciferol: The confirmation test of the raw material for
manufacturing of vitamin D powder is applied mutatis mutandis.
iii. dl-α-tocopherol acetate: 5 µL of the sample solution for thin layer chromatographobtained by the assay is measured and spotted on a thin layer plate. 5 µL of thestandard solution for thin layer chromatograph is spotted in parallel on the same thinlayer plate. Then, they are developed to approximately 10 cm with the developing
solvent, a mixed solution of n-hexane and ethyl acetate (4:1), sprayed with dilute ferricchloride test solution, and then sprayed with α,α'-dipyridyl test solution, and the spots

672

Provisional Translation from Japanese Originalof tocopherol obtained from the sample and standard solutions are both red and their
Rf values are equal.
Assay:
i. Vitamin A: It tested by Method No. 2 of the vitamin A assay.
ii. Cholecalciferol or ergocalciferol: It tested by method of the vitamin D assay.
iii. dl-α-tocopherol acetate: The amount of this product containing approximately 0.02 gof dl-α-tocopherol acetate (C31H52O3) is weighted to three significant digits and thevalue is recorded. It is saponified, extracted, and dehydrated by the vitamin A assay.
The ether extract is distilled off under reduced pressure in water at 45 °C, 2 mL ofacetone is measured using a volumetric pipette, and it is added in the residue anddissolved. This is used as a sample solution for thin layer chromatograph. Separately,
approximately 0.02 g of dl-α-tocopherol acetate for assay is weighed to the digits of
0.1 mg and the value is recorded. It is subjected to the same procedure of the
preparation of a sample solution and used as a standard solution of thin layer
chromatograph. 0.2 mL each of the sample and standard solutions of thin layer
chromatograph are measured using a volumetric pipette and spotted on a thin layer
plate prepared using silica gel for thin-layer chromatography (with a fluorescentagent). Then, they are developed to approximately 15 cm with the developing solvent,a mixed solution of n-hexane and ethyl acetate (4:1), and the thin layer plate is air
dried and irradiated with ultraviolet light (dominant wavelength: 254 nm). The parts of
tocopherol obtained from the sample and standard solutions are collected by scratchingwith a stainless microspatula, and placed in a 50 mL beaker. It is added with a little
ethanol, shaken, and allowed to stand, and the supernatant is filtered with a glass filter.
Extraction is performed by repeating the same procedure. The extract is transferred toa 100 mL volumetric flask and added with 100 mL of ethanol to the graduation line to
make 100 mL. This is used as a sample and standard solutions. 10 mL each of thesample and standard solutions are measured using a volumetric pipette, transferred to
25 mL volumetric flasks, added with 1 mL of dilute ferric chloride test solution and 1
mL of α,α'-dipyridyl test solution, and added with ethanol to the graduation line to
make 25 mL. Separately, 10 mL of ethanol is measured using a volumetric pipette andsubjected to the same procedure to prepare a control solution. Then, the absorbances
AT and AS at the wavelength 520 nm are measured.
Amount of dl-α-tocopherol acetate (mg)
= Amount of dl-α-tocopherol acetate for assay (mg) ×

673

AT
AS

Provisional Translation from Japanese Original(b) Standard of storage method
It shall be stored in a lightproof airtight container making it almost full or with nitrogen
replaced with air.
B. Preparation (Part 2: powder)(a) Compositional standards
This is powder or particles, which is a mixture of the raw material for manufacturing or
preparations of vitamin A oil or vitamin A powder, the raw material for manufacturing or
preparations of cholecalciferol, ergocalciferol, vitamin D3 oil or vitamin D powder, and theraw material for manufacturing or preparations of dl-α-tocopherol acetate or vitamin E
powder in fillers.
Content: The assay shows that this product contains vitamin A (retinol) corresponding to
90~130 % of the unit on the label, cholecalciferol (C27H44O) or ergocalciferol (C28H44O)
corresponding to 90~130 % of the unit on the label, and dl-α-tocopherol acetate(C31H52O3) corresponding to 90~120 % of the unit on the label.
Confirmation test:
i. Vitamin A: The confirmation test of the raw material for manufacturing of vitamin A
powder is applied mutatis mutandis.
ii. Cholecalciferol or ergocalciferol: The confirmation test of the raw material for
manufacturing of vitamin D powder is applied mutatis mutandis.
iii. dl-α-tocopherol acetate: The confirmation test iii.

of the preparation (Part 1: liquid)of vitamin ADE is applied mutatis mutandis.
Assay:
i. Vitamin A: The amount of this product containing vitamin A 500 units or more and 1 gor less of fat and oil is weighed to three significant digits and the value is recorded. It
is placed in a flask, added with 2 mL of water, heated for a while with shaking, thenadded with 30 ml of aldehyde-free ethanol and 1 ml of ethanol solution of pyrogallol(1 → 10), and from here the test by Method No. 2 of the vitamin A assay is performed.
ii. Cholecalciferol or ergocalciferol: It tested by method of the vitamin D assay.
iii. dl-α-tocopherol acetate: The assay of Vitamin ADE preparation (Part 1: liquid) is
applied mutatis mutandis.
(b) Standard of storage method
It shall be stored in a lightproof capped container.

674