SRC TR

- 10

- 114

HPV4 CHEMICAL DATA ADEQUA CY REVIEW

Glycine, N

- (carboxymethyl)

- , sodium salt , CAS No. 928

- 72

- 3

Prepared by:

Christopher Janson

, Justine Von Runnen

, Christine Carnegie, Julie Ramage, Teresa Manyin, and

Joan D. Garey

Chemica l, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007.0.009.002

Submitted to:

OPPT/RAD/HPVCB (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Elizabeth Margosches

, Alternate Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

August 18 , 2010HPV4 Chemical Data Adequacy Review

Glycine, N

- (carboxymethyl)

- , sodium salt , CAS No. 9 28

- 72

- 3

August 2010

Submitted to:

High Production Volume Chemicals Branch

Risk Assessment Division

Office of Pollution Prevention and Toxics

Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks

1200 Pennsylvania Avenue, NW

Washington, DC 20460

- 0001

Prepared By:

SRC , Inc.

Chemical, Biological and Environmental Center

2451 Crystal Drive, Suite 804

Arlington, VA 22202Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

1 A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A

- 1. Source material used for assessment

For this unsponsored chemical, a comprehensive literature search was performed to

identify available toxicity data for the following SIDS endpoints:

Acute toxicity to fish

Acute toxicity to Daphnia

Toxicity to aquatic plants

Acute (oral, dermal, inhala tion) toxicity

Repeated

- dose toxicity

Reproductive toxicity

Developmental toxicity

Gene mutation

Chromosomal aberrations

Studies relevant to non

- SIDS endpoints (e.g., eye and skin irritation; neurotoxicity) conducted

via oral, inhalation, or dermal routes were also identified through literature searching.

The following sources were searched for toxicity data:

PubMed

- B ibliographic database that contains references to journal articles in the life

sciences with a concentration on biomedicine

T OXNET

- N ati onal L ibrary of M edicine databases on toxicology, hazardous chemicals,

environmental health, and toxic releases; includes:

**•** TOXLINE with BIOSIS and NTIS ;

**•** Development and Reproductive Toxicology File (DART) ;

**•** Chemical Carcinogenesis Research Information Sy stem (CCRIS)

- Carcinogenicity and mutagenicity test results for over 8000

**•** chemicals) ;

**•** Hazardous Substances Data Bank (HSDB)

- Comprehensive, peer

reviewed toxicology data for about 5000 chemicals ;

**•** Genetic Toxicology Data Bank (GENE

- TOX)

- Peer

- reviewed genetic

toxicology test data for over 3000 chemicals.

TSCATS

- U npublished technical reports submitted by industry to the EPA under the

Toxic Substances Control Act]

Registry of toxic effects of chemical substances (RTECs)

Environmental Fate Database ( EFDB)

ECOTOX

- EPA’s peer

- reviewed ecotoxicology databaseDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

2 EPA’s Integrated Risk Information System (IRIS) website

Agency for Toxic Substances and Disease Registry (ATSDR) website

National Toxicology Program (NTP) website

International Program on Chemic al Safety Concise International Chemical Assessment

Documents (IPCS INC HE M or CICADs)

European Chemical Substances Information System (ESIS)

- source of IUCLID Datasets

Any available IUCLID Datasets found through the ESIS website and robust summaries

su bmitted to EPA by former sponsors on the assigned chemicals were retrieved. Original

sources were obtained for any relevant toxicity data found for the assigned chemicals.

While IUCLID D ataset s sometimes contained studies of interest, certain study

des criptions contained insufficient detail to assess adequacy. In these cases, the primary source

of the study, if available and accessible, was obtain ed and review ed.

A

- 2. Evaluating data adequacy

Using standard OECD testing guidelines and professiona l judgment as guides, available

toxicity studies for the unsponsored chemical were reviewed for adequacy. Summaries were

prepared for all relevant data found, even if little or no study detail was available. Each study

was summarized according to a standa rd format and assigned a Klimisch reliability code; all

study summaries for the chemical were combined into a single summary document. For studies

already assigned Klimisch codes (e.g., studies summarized in IUCLID datasets), an explanation

was provided i f the reviewer’s assessment of data adequacy differed from the pre

- assigned

Klimisch code. A table summarizing data availability/adequacy for each SIDS endpoint is

included at the end of the summary document.

B. SPECIFIC INFORMATION ON THE AVAILABLE TOXICITY DATA

The available data for each of the SIDS toxicity endpoints are evaluated on the following

pages. Studies for a common endpoint are grouped together in the document and an endpoint

adequacy assessment is provided near the top of the first p age of the summaries for that

endpoint. If the data available for the endpoint is determined to be inadequate, a basis for that

conclusion is supplied. NOTE: If no relevant studies were identified for the chemical , only the

data availability/adequacy su mmary table was completed for the report

.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

3 B

- 1. TOXICITY TO AQUATIC VERTEBRATES

Summary of endpoint: Adequate

Study 1: Predicted toxicity to fish

Title: ECOSAR

- estimated toxicity to fish

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Disodium Iminodiacetic Acid (DSIDA), CAS No. 928

- 72

- 3

Remarks: N/A

METHOD:

Method/guideline followed: Estimated by E COSAR version 1.00

Molecular weight = 177.07

Log Kow =

- 7.51

Water solubility = 1E +0 0 6 mg/L

Test type: Acute 96

- hour toxicity estimate

GLP compliant?: N/A

Year study was performed: N/A

Test species: Fish

Analytical monitoring: N/A

Exposure duration: 96 hours

Concentrations tested: N/A

Test method/conditions remarks: N/A

RESULTS

Endpoint value(s): 96h LC50 = 1.64 e+00 7 mg/L

Results remarks:

96

- hour LC50 was estimated from ECOSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

4 CONCLUSIONS:

The predicted 96h LC50 for fish is 1.64 e+00 7 mg/L

.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions

– endpoint value estima ted using a modeling program

.

REFERENCE

ECOSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

5 B

- 2. TOXICITY TO AQUATIC INVERTEBRATES

Summary of endpoint: Adequate

Study 1: Predicted toxicity to daphnids

Title: ECOSAR

- estimated toxicity to daphnids

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Disodium Iminodiacetic Acid (DSIDA), CAS No. 928

- 72

- 3

Remarks: N/A

METHOD:

Method/guideline followed: Estimated by ECOSAR version 1.00

Molecular weight = 177.07

Log Kow =

- 7.5 1

Water solubility = 1E +00 6 mg/L

Test type: Acute 48

- hour toxicity estimate

GLP compliant?: N/A

Year study was performed: N/A

Test species: Daphnid s

Analytical monitoring: N/A

Exposure duration: 48 hours

Concentrations tested: N/A

Test method/condi tions remarks: N/A

RESULTS

Endpoint value(s): 48h LC50 = 2.32e+005 mg/L

Results remarks:

48

- hour LC50 was estimated from E COSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

6 CONCLUSIONS

The predicted 48 h LC50 for daphnids is 2.32e+005 mg/L

.

STUDY RELIABILITY

[ 2 ] Reliable with restr ictions

– endpoint value estimated using a modeling program

.

REFERENCE

E COSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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- 3

7 B

- 3. TOXICITY TO AQUATIC PLANTS

Summary of endpoint: Adequate

Study 1: Predicted toxicity to green algae

Title: ECOSAR

- estimated toxicity to green algae

Thi s study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Disodium Iminodiacetic Acid (DSIDA), CAS No. 928

- 72

- 3

Remarks: N/A

METHOD:

Method/guideline followed: Estimated by E COSAR version 1.00

Molecular we ight = 177.07

Log Kow =

- 7.51

Water solubility = 1E +00 6 mg/L

Test type: Acute 96

- hour toxicity estimate

GLP compliant?: N/A

Year study was performed: N/A

Test species: Green algae

Exposure duration: 96 hours

Concentrations tested: N/A

Test method/co nditions remarks: N/A

RESULTS

Endpoint value(s): 96h EC50 = 4527.094 mg/L

Results remarks:

96

- hour EC50 was estimated from E COSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

8 CONCLUSIONS

The predicted 96 h E C50 for green algae is 4527.094 mg/L

.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions

– endpoint value estimated using a modeling program

.

REFERENCE

E COSAR version 1.00Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

9 B

- 4. ACUTE TOXICITY

Summary of endpoint: Adequate, based on a weight

- of

- evidence approach using studies from

secondary sources with many study details missing. The studies summarized here demonstrate

low toxicity of glycine and g lycine, N

- (carboxymethyl)

- , sodium salt

.

Study 1: Acute oral toxicity study in rats

Title: Disodium Iminodiacetic Acid (DSIDA): Acute Oral Toxicity (Albino Rats)

The study i s: Adequate Not Adequate

TEST SUBSTANCE

Identity: Disodium Iminodiacetic Acid (DSIDA), CAS No. 928

- 72

- 3

Remarks: ID No. 3

- 18

- 85

- 1500

METHOD

Method/Guideline followed: Not stated

Test Type: Acute oral toxi city

GLP (Y/N): Yes

Year study performed: 1985

Species/Strain: Sprague

- Dawley albino rats

Sex (males/females/both): Both

Number of animals per dose: 5/sex/dose

Age and body weight range of animals used: Initial weight: 190

- 200 g

Route of admini stration: Gavage

Vehicle: None

Dosing regimen : Animals received a single oral gavage dose of undiluted test substance,

warmed to 58 deg C. Animals were observed for 14 days post

- dosing.

Doses: 2000, 2510, 3160, 3980 or 5010 mg/kg

- bw

Statistical meth ods used: Not statedDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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10 Remarks: None

RESULTS

Male mortalities were observed in all but the low

- dose group (mortalities were 1/5, 2/5, 5/5, and

5/5 at 2510, 3160, 3980 and 5010 mg/kg

- bw respectively ). Female mortalities were observed at

all dosage levels (mortalities were 1/5, 2/5, 3/5, 3/5, and 5/5 at 2000, 2510, 3160, 3980 and 5010

mg/kg

- bw respectively ). Signs of toxicity observed in males included lethargy (lasting up to

nine days), diarrhea (in one instance), increasing weakness, collapse and death. Gross necropsy

of males revealed lung hyperemia (hemorrhagic in most instances), hemorrhagic liver

(discolored in most instances), kidney and spleen discoloration (in most instances), and acute

gastrointestinal inflammation. Toxic signs observed in fema les included lethargy (lasting up to

14 days), increasing weakness, collapse and death. Gross necropsy of females revealed

hemorrhagic lung, hemorrhagic liver (discolored in most instances), kidney discoloration, spleen

discoloration, and acute gastrointe stinal inflammation.

CONCLUSIONS

The male rat LD50 was 3310 mg/kg (2850

- 3870 mg/kg); the female rat LD50 was 2810 mg/kg

(2250

- 3510 mg/kg). The LD50 for males and females combined was 3160 mg/kg (2250

- 3630

mg/kg).

RELIABILITY CODE

[ 2 ] Reliable with res trictions.

REFERENCE

Disodium Iminodiacetic Acid (DSIDA): Acute Oral Toxicity (Albino Rats). Study Number:

YO

- 85

- 073. Obtained from 8d 8607

- 0000045 Monsanto.pdf: page 1249. Received November

20, 2006.

Disk 3, p. 5269

- 5270Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

11 Study 2: Acute dermal toxici ty in rabbits

Title: Disodium Iminodiacetic Acid (DSIDA): Acute Dermal Toxicity (Albino Rabbits)

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Disodium Iminodiacetic Acid (DSIDA), CAS No. 928

- 72

- 3

Remarks: disodium imino diacetic acid, ID No. 3

- 18

- 85

- 1500

METHOD

Method/Guideline followed: Not stated

Test Type: Acute dermal toxicity

GLP (Y/N): Yes

Year study performed: 1985

Species/Strain: New Zealand White rabbits

Sex: Both

Number of animals per dose: 2/sex/dose

Age and body weight range of animals used: Initial average body weight: 2.2

- 2.3 kg

Route of administration: Dermal

Vehicle: None

Dosing regimen : Application of the undiluted (warmed to 50 deg C) test substance was mad e

on intact skin. Exposure, which lasted 24 hours, was followed by 14 days of observation.

Doses: 5010 mg/kg

- bw

Statistical methods used: Not stated

Remarks: None

RESULTS

No mortalities or toxic signs were observed. Viscera appeared normal.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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12 CONCLU SIONS

The dermal LD50 was >5010 mg/kg

- bw.

RELIABILITY CODE

[ 2 ] Reliable with restrictions.

REFERENCES

Disodium Iminodiacetic Acid (DSIDA): Acute Dermal Toxicity (Albino Rabbits). Study

Number: YO

- 85

- 073. Obtained from 8d 8607

- 0000045 Monsanto.pdf: p age 1250. Received

November 20, 2006

Disk 3, p. 5269, 5271Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

13 Study 3 : Acute oral toxicity study in rats

Title: Acute oral toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to m issing critical study information; secondary

source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline followed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test spec ies: Rat, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: Oral

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoin t value(s): 7930 mg/kg bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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14 CONCLUSIONS

The acute oral LD50 in rats is 7930 mg/kg bw

.

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Yaesu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77

Lewis, R.J., Food Additives Handbook p. 235

- 236 (1989); FASP Study No. 90Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

15 Study 4: Acute oral toxicity study in mice

Title: Acute oral toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; secondary

source.

TE ST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline followed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test species: Mouse, species/strain not provided

Number of anim als/sex/dose: No data

Route of administration: Oral

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 4920 mg/kg bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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16 CONCLUSIONS

The acute oral LD50 in rats is 4920 mg/kg bw

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chir yo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Yaesu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

17 Study 5: Acute s.c. toxicity study in rats

Title: Acute s.c. toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; non

- standard

exposure route; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline fol lowed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test species: Rat, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: s.c.

Vehicle: No data

Doses/concentrations tested: No d ata

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 5200 mg/kg bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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18 CONCLUSIONS

The acute s.c. LD50 in rats is 5200 mg/kg bw

STUDY RELIABILITY

[4] Not assignable

– no st udy detail provided; non

- standard exposure route; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Yaes u, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77

Lewis, R.J., Food Additives Handbook p. 235

- 236 (1989); FASP Study No. 90Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

19 Study 6: Acute i.p. toxicity study in mice

Title: Acute i.p. toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; non

- standard

exposure route; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline fol lowed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test species: Mouse, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: i.p.

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 4450 mg/kg bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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20 CONCLUSIONS

The acute i.p. LD50 in mice is 4450 mg/kg bw

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; non

- standard exposure route; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Ya esu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

21 Study 7: Acute s.c. toxicity study in mice

Title: Acute s.c. toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; non

- standard

exposure route; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline followed: No data

Test type: LD50

GLP compliant?: No data

Year study perfor med: No data

Test species: Mouse, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: s.c.

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 5060 mg/kg

- bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

22 CONCLUSIONS

The acute s.c. LD50 in mice is 5060 mg/kg bw

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; non

- standard exposure route; secondary source

.

REFE RENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Yaesu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

23 Study 8 : Acute i. v. toxicity study in rats

Title: Acute i.v. toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; non

- standard

exposure route; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline followed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test species: Rat, species/strain not provided

Number of an imals/sex/dose: No data

Route of administration: i.v.

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 2600 mg/kg

- bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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24 CONCLUSIONS

The acute i.v. LD50 in rats is 2600 mg/kg bw

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; non

- standard exposure route; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2

- 5

- 13, Yaesu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

25 Study 9: Acute i.v. toxicity study in mice

Title: Acute i.v. toxicity

The study is: Adequate Not Adequate

This study was considered inadequate due to missing critical study information; non

- standard

exposure route; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No dat a

METHOD

Method/guideline followed: No data

Test type: LD50

GLP compliant?: No data

Year study performed: No data

Test species: Mouse, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: i.v.

Vehicle: No data

Doses/concentrations tested: No data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 2370 mg/kg

- bw

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

26 CONCLUSIONS

The acute i.v. LD50 in mice is 2370 mg/kg bw

STUDY RELIAB ILITY

[4] Not assignable

– no study detail provided; non

- standard exposure route; secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Yachds Yakuri to Chiryo. Pharmacology and Theraputics (Raifu Sa iensu Shuppan K. K., 2

- 5

- 13, Yaesu, Chuo

- ku, Tokyo 104, Japan. V.1

– 1972

- 5, 1502, 77Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

27 Study 9: Acute i.v. toxicity study in the cat

Title: Acute i.v. toxicity

The study is: Adequate Not Adequate

This study was co nsidered inadequate due to missing critical study information; non

- standard

exposure route; non

- standard species tested; secondary source.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: No data

METHOD

Method/guideline followed: No d ata

Test type: LDL0

GLP compliant?: No data

Year study performed: No data

Test species: Cat, species/strain not provided

Number of animals/sex/dose: No data

Route of administration: i.v.

Vehicle: No data

Doses/concentrations tested: No data

Statis tical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 3 g/kg

Results remarks: No dataDraft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

28 CONCLUSIONS

The acute i.v. LDL0 in cats is 3 g/kg

STUDY RELIABILITY

[4] Not assignable

– no study detail provided; non

- standard species and exposure route;

secondary source

.

REFERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000

Journal of the American Pharmaceutical Association Scientific Edition (Washington DC) V.29

- 49, 1940

- 60. 31, 3 06, 42Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

29 B

- 5: REPEATED

- DOSE TOXICITY

Summary of endpoint: Inadequate

Study 1: Repeated

- dose dietary toxicity study in rats

Title: Pteroylglutamic Acid, Ascorbic Acid, and Injectable Liver Extract on Dietary Glycine

Toxicity in the Rat

The study is: Adequate Not Adequate

This study is not considered adequate to fulfill the repeated

- dose toxicity endpoint because it is

not a standard protocol study, only one dose level was tested and LOAEL/NOAEL values were

not repo rted. Additionally, limited study details were provided.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: P urity not reported

METHOD

Method/guideline followed: None stated

Test type: S hort term dietary study

GLP compliant?: Not stat ed

Year study performed: 1949

Test species: Sprague

- Dawley weanling rats

Number of animals/sex/dose: 4/sex/group

Route of administration: Oral, diet

Vehicle: F ood

Exposure duration: 46 days

Doses/concentrations tested: 0% (group I) or 10% (groups II

– VI)

Statistical methods used: The differences in weight gains among the various groups were

statistically treated by the analysis of variance method.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

30 Test method/conditions remarks: Six groups of weanling Sprague

- Dawley rats, 4 males and 4

females in each group, were fed a standard purified diet, and a similar diet containing 10%

glycine replacing an equal amount of sucrose. Groups of rats were given the glycine

- containing

diet supplemented respectively with PGA, Injectable liver extract, PGA plus liver extract, and

ascorbic acid. The rats were fed ad libitum and food intake was measured. Complete blood

counts were made on the 37 th and 46 th days of the experiment.

Composition of Experimental Diets

Dietary component Groups

I II III IV V VI

Casein, % 18 18 18 18 18 18

Sucrose, % 75 65 65 65 65 65

Crisco, % 3 3 3 3 3 3

Cod liver oil, % 2 2 2 2 2 2

Salt mix, % 2 2 2 2 2 2

Glycine, % 0 10 10 10 10 10

PGA

— mg/100 g 0 0 0.5 0 0.5 0

Liver extr.

— cc/kilo 0 0 0 8 8 0

Ascorbic acid

— mg/100 g 0 0 0 0 0 100

RESULTS

Endpoint value(s): Not reported

Results remarks: It may be observed that the inclusion of 10% glycine in the purified diet

resulted in a greatly decreased rate of growth. PGA markedly improved the growth of rats

receiving the high glycine diet. The addition of liver extract to the high glycine diet resulted in

some growth improvement but less than that produced by PGA. The liver extract used was

assayed for PGA and found to contain 2.62 y per cc; this amounted to approximatel y 0.2 y of

PGA per rat per day. This and the fact that the group receiving PGA plus liver extract grew no

faster than those receiving PGA alone would indicated that some of the growth improvement

resulting from the liver extract may have been due to its c ontent o f PGA.

Ascorbic acid had little effect on the growth of the rats. The difference in rate of gain between

the group receiving ascorbic acid and the group receiving high glycine alone was not statistically

significant. Weight gains per gram of f ood eaten were calculated for the various groups and the

average values were: control 0.295, high glycine 0.152, high glycine plus PGA 0.282, high

glycine plus liver extract 0.205, high glycine plus PGA plus liver extract 0.291, and high glycine

plus ascor bic acid 0.213. With the exception of reversal between the ascorbic acid and liver

extract groups, efficiency of gain ranked the same in the various groups as did total weight

changes.

The feeding of the high glycine diet did not produced a significant leucopenia in these rats when

compared o the controls; however, addition of PGA to this diet did elevate the white cell count.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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31 Addition of liver extract and liver extract plus PGA to the high glycine diet resulted in an

increased white count in male rats but was without effect in the females.

Although the red cell counts of rats receiving the high glycine diet were lower than the control

group, none of the supplements tested were effective in restoring the counts to the level of the

controls. Hemoglob in values were also reduced by the high glycine diet and were improved only

by the addition of the combination of PGA plus liver extract or by ascorbic acid. There were no

consistent differences in hematocrit or differential white cell count among the var ious groups.

The rats receiving ascorbic acid were found to have a higher mean corpuscular volume than any

of the other groups. It may also be noted that rats receiving ascorbic acid had the lowest red

count of any of the groups, but had a higher hemoglo bin level than any of the others except the

control group and the group receiving liver extract plus PGA.

There is not sufficient evidence available at present to permit an explanation of the growth

promoting effect of PGA when added to a high glycine die t for rats.

CONCLUSIONS

Overall, the growth of rats receiving the high glycine diet was greatly reduced as compared to

the control group. Supplementation of the high glycine diet with PGA and with PGA plus liver

extract resulted in a marked improvemen t in growth rate; liver extract alone was less effective;

and ascorbic acid did not significantly improve rate of growth of rats receiving the 10% glycine

diet. Rats receiving the high glycine diet did not develop a marked leucopenia when compared

with co ntrol rats.

STUDY RELIABILITY

[4] Not assignable

– not a standard protocol study, limited study details were provided, only one

dose level was tested and LOAEL/NOAEL values were not reported

.

REFERENCE

Dinning et al . “Pteroylglutamic Acid, Ascorbic A cid, and Injectable Liver Extract on Dietary

Glycine Toxicity in the Rat (17400)”. Proc. Soc. Exp. Biol. Med. 72: 262

- 264 (1949). FDA

PAFA profile for Glycine (7/29/2010). FASP Study No. 93.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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32 B

- 6: CARCINOGENICITY

Study 1: Chronic toxicity/carcinogenici ty study in rats

Title: Carcinogenicity study of glycine in Fischer 344 rats

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: Lot No. 001103, produced by Yuhkigosei Yakuhin Kogyo Co. as a white crystalline

powder with a sweet taste

MET HOD

Method/guideline followed: United States National Cancer Institute Guidelines for Carcinogen

Bioassay in Small Rodents (NCI, Bethesda, MD. 1974)

Test type: Chronic toxicity/carcinogenicity

GLP compliant?: Not stated

Year study performed: 1994

Tes t species: Fischer (344 DuCrj) rats obtained from Charles River Co. Japan, Inc. Atsugi. Rats

used in this study were 6 weeks old at the start of the study.

Number of animals/sex/dose: 50/sex/dose

Route of administration: oral, drinking water

Vehicle: D rinking water

Exposure duration: 108 weeks

Doses/concentrations tested: 0%, 2.5% or 5.0%

Statistical methods used: Effective numbers of rats included both those killed at week 108 and

those which died with tumors earlier. The data were evaluated by th e Wilcoxon t

- test or the x 2

test.

Test method/conditions remarks: The rats were divided into three groups of 50 males and

females each when 6 weeks old: group I was given 5% GL and group II 2.5% GL in the drinking

water, while the control (group III) rece ived the drinking water without GL. The feed and

drinking water, with or without GL, were freely available. The rats were weighed every one or

two weeks and their diet consumption recorded every 4 weeks. All survivors were killed at week

108, blood samp les being then taken from all animals of both sexes in each group forDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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33 measurement of white blood cells, red blood cells, hemoglobin (Hb), hematocrit (Ht), GOT,

GPT, LDH, CPK, BUN, CRE, and Urea at the Ina Clinical Laboratory Inc., Iga Ueno.

Complete auto psy was performed on all animals, including those that died or were sacrificed

upon becoming moribund during the experiment. All organs were carefully examined and

samples were fixed in 10% buffered neutral formalin, embedded in paraffin, and routinely

st ained with hematoxylin and eosin. Histological analysis of tumors in renal pelvis was

conducted. Effective numbers of rats included both those killed at week 108 and those which

died with tumors earlier.

RESULTS

Results: Growth curves, mortality and d iet consumption: A slight dose

- dependent inhibition of

mean body weight gain was observed in both sexes. At the end of the administration period, the

numbers of surviving male animals were 40 in the 5.0% group, 45 in the 2.5% group and 40 in

the control g roup, and for females 31, 46, and 41, respectively.

Urinalysis: The pH of urine samples from 6.0 to 7.5 for all female groups and male rats treated

with 5.0 or 2.5% GL and from 6.5 to 7.0 for control males. There were no significant differences

in urinal ysis data between treated and control rats.

Blood analysis: The Hb in male rats given 5.0% GL was significantly higher than that in control

male rats but that in the female case 5.0% GL administration was associated with a significant

reduction. The Ht in female rats given 5.0% GL was also significantly lower than in control

female rats. The CPK values for both sexes given 5.0% or 2.5% GL were significantly lower

than the respective control values and the BUN in both male and females on 5.0% GL

significan tly increased.

Final body and organ weights, and total glycine intake: The final body weights of both sexes

given 5.0% GL were significantly lower than those of controls. However, there were no

significant intergroup differences in relative organ weigh ts. The total glycine intake was directly

related to the dietary dose in both sexes.

Tumor incidence: For the males, tumors were found in 90% of the 5.0% GL group, in 97% of

the 2.5% and in 90% of the controls, while the corresponding figures for fema les were 59, 65,

and 39%. These high proportions of tumor

- bearing rats were mainly due to testis, thyroid,

pituitary gland, pancreas, and uterine lesions. Kidney papillomas were found in 8% of the 2.5%

GL and 6% of the 5.0% GL group females, but not in o ther groups. A renal cell tumor was seen

in one of 40 male rats fed on 5.0% GL. A transitional cell tumor of the pelvis was seen in one of

40 male control rats.

Non

– malignancy histological findings for the urinary system: The incidences of calcifica tion in

kidney were 39, 32, and 42% in females of the 0, 2.5 and 5% GL groups, respectively. The

incidences of papillary necrosis in male rats fed on 2.5 and 5.0% GL were 4 and 7%, while the

corresponding figures for females were 30 and 32%. Hyperplasia of transitional cells in renal

pelvis was seen in 2 of 46 female rats fed 2.5% GL.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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34 CONCLUSIONS

Glycine was carcinogenic to rats in this study.

STUDY RELIABILITY

[ 1 ] Reliable with out restrictions.

REFERENCE

Kitahori et al

., 1994

. Carcinogenicity Stud y of Glycine in Fischer 344 Rats. J Toxicol Pathol 7:

471

- 480. FDA PAFA profile for Glycine (7/29/2010). FASP Study No. 98.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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35 B

- 7: GENE MUTATION STUDIES

Summary of endpoint: Adequate

Study 1. In vitro gene mutation study in Salmonella

Title: Gen etic Toxicity in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: Not stated

METHOD

Method/guideline followed: Preincubation protocol

Test type: Ames Salm onella assay

GLP compliant?: Yes

Year study performed: 1979

Test species: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537

Metabolic activation: With and without S9 ( Aroclor 1254

- induced male Sprague

- Dawley rat or

Syrian hamster liver)

C oncentrations tested: 0, 100, 333, 1000, 3333 and 10,000 μg/plate

Statistical methods used: Not stated

Test method/conditions remarks: Salmonella typhimurium strains TA98, TA100, TA1535 and

TA1537 were exposed to glycine in water at 0, 100, 333, 1000, 33 33 and 10,000 μg/plate

without and without metabolic activation. A positive control was tested concurrently.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): With metabolic activation, toxicity was observed in

strain TA153 7 at a concentration of 3333 μg/plate only.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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36 Results remarks: Not stated

CONCLUSIONS

Glycine was not mutagenic in this assay.

STUDY RELIABILITY

[1] Reliable without r estrictions

.

REFERENCE

NTP (1979). Salmonella (152901). Citation: Haworth, S., La wlor, T., Mortelmans, K., Speck,

W., and Zeiger, E. Salmonella mutagenicity test results for 250 chemicals Environ. Mutagen.

Vol. 5 (Suppl 1) (1983) 3

- 142.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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37 Study 2: In vitro gene mutation study in Salmonella

Title: Genetic Toxicity in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: Not stated

METHOD

Method/guideline followed: Preincubation protocol

Test type: Ames Salmonella assay

GLP compliant?: Yes

Year study performed: 1979

Test species: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537

Metabolic activation: With and without S9 ( Aroclor 1254

- induced male Sprague

- Dawley rat or

Syrian hamster liver)

Concentrations tested: 0, 10, 33, 1 00, 333 and 1000 μg/plate

Statistical methods used: Not stated

Test method/conditions remarks: Salmonella typhimurium strains TA98, TA100, TA1535 and

TA1537 were exposed to glycine in water at 0, 10, 33, 100, 333 and 1000 μg/plate without and

without met abolic activation. A positive control was tested concurrently.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): No cytotoxicity was observed.

Results remarks: Not stated

CONCLUSIONS

Glycine was not mutagenic in this a ssay.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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38 STUDY RELIABILITY

[1] Reliable without r estrictions

.

REFERENCE

NTP (1979). Salmonella (709932). Citation: Haworth, S., Lawlor, T., Mortelmans, K., Speck,

W., and Zeiger, E. Salmonella mutagenicity test results for 250 chemicals Environ. Mutagen

. Vol. 5 (Suppl 1) (1983) 3

- 142.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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39 Study 3: In vitro gene mutation in Escherichia coli

Title: Mutagenesis by normal metabolites in Escherichia coli : phenylalanine mutagenesis is

dependent on error

- prone DNA repair

The study is: Adequat e Not Adequate

This study was not considered adequate to fulfill this endpoint because the article and main study

focused on the mutagenicity of phenylalanine, not glycine. Additionally, only one test substance

concentration was tested in the absence of metabolic activation.

TEST SUBSTANCE

Identity: L

- glycine, CAS No. 56

- 40

- 6

Remarks: Eastman Organic

METHOD

Method/guideline followed: Not stated

Test type: Mutagenicity in Escherichia coli

GLP compliant?: Not stated

Year stud y performed: 1985

Test species: Escherichia coli strain uvrB umuC

Metabolic activation: No

Concentrations tested: 2 mM

Statistical methods used: Not stated

Test method/conditions remarks: P la te assa y for mutagenesis : Logarithmic

- phase cells were

prepa red by diluting overnight cultures 1:50 into warm SMM 1 , and shaking the cultures at 37°C

until a concentration of 4 x 10 8 colony

- forming units (CFU) per ml was attained. Cells were

pelleted by centrifugation, washed once, and resuspended in PB (phosphate buffer) at 1.5 x 10 8

CFU/ml. Cells (0.1 ml) were spread on Glu

- 600 plates (lactose plates containing a growth

limiting amount of glucose , i.e., 600 μg/mL) ± the test compound at 2 mM and Glu

- 0 plates.

Plates were incubated for 3 days at 37°C before scorin g Lac + mutants. To determine the relative

mutagenesis, the mean number of mutants on 5 Glu

- 0 plates (i.e., preexisting mutants) was

subtracted from the mean number of mutants on 10 Glu

- 600 plates (i.e., plate mutants) and from

the mean number of mutants o n 5 Glu

- 600 plates containing the test compound (i.e., plate plus

induced mutants). Then the ratio (test/control) was determined. The mean numbers of

pree xisting mutants per plate were 1 (wi l d

- type), 15 ( u v rB ), and 0 ( uvrB umuC and u vrB l exA ),Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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40 while the mea n number of plate mutants per control plate were 60(wild

- type), 355( u v rB ),

2 1 ( u v rB umuC ), and 20( uvrB l exA ). For Glu

- 600 control plates, the total L ac cells per plate was

3 .4 X 10 10 for both the uvrB and uvrB umuC strains.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not stated

Results remarks: Glycine had no significant effect on Lac reversion in a uvrB umuC strain

CONCLUSIONS

Glycine was not mutagenic in this assay.

STUDY RELIABILITY

[3] Not reliable

.

REFERE NCE

Sargentini, N.J., and Smith, K.C. "Mutagenesis by normal metabolites in Escherichia coli:

phenylalanine mutagenesis is depended on error

- prone DNA repair", 1986. Mutation Research,

161: 113

- 118. FASP Study No. 83Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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41 Study 4: In vitro gene mutation i n Salmonella

Title: Mutagenicity of heated solutions of Vitamin K 1 with amino compounds

The study is: Adequate Not Adequate

This study was not considered adequate to fulfill this endpoint because only one Salmonella

strain was tested in the presence of metabolic activation, the test substance was a mixture with

vitamin K 1 , and limited study details were provided.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: G ene mutation in Salmonella

GLP compliant?: Not stated

Year study performed: 1983

Test species: Salmonella strain TA98

Metabolic activation: W ith S9

Concentrations tested: 500 μmoles with aliquots of 2, 10 and 50 μmol es assayed for

mutagenicity.

Statistical methods used: Not stated

Test method/conditions remarks: Since vitamin K 1 reacted with creatine to yield exceptionally

high mutagenic activity, further reactions with this compound were carried out against cre atinine,

arginine, threonine and glycine.

Heat induced reactions between the amino compounds and various fat constituents were carried

out using a model system which has been used to study browning reactions. In this procedure

the reactants are heated in ethylene glycol, a high boiling neutral solvent which allowed the

reactions to take place under homogeneous conditions while minimizing the loss of mutagens

due to volatilization. 500 μmoles each of the reactants were added to ethylene glycol: water (9 :

1) in a 125

- ml Erlenmeyer flask and heated for 90 min on a hot plate kept at 200°C. After

cooling, the reaction mixture was diluted with 200 ml 1 N HCl. It was extracted once with 100Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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42 ml methylene chloride and the organic layer discarded. The aqueous layer was adjusted to pH 11

with sodium hydroxide and the basic mutagens were extracted twice with 100

- ml aliquots of

methylene chloride. After removal of traces of moisture by filtering through anhydrous sodium

sulfate, the methylene chloride extracts wer e taken to dryness, under vacuum at 40°C. The

residue was taken up in DMSO and aliquots representing 2, 10 and 50 μmoles of the original

reactant(s) were assayed for mutagenic activity against S. typhimurium strain TA98 in the

presence of Aroclor

- induced r at

- liver microsome (S9) preparation according to the standard

Ames procedure.

RESULTS

Genotoxicity result: Positive

Results remarks: While glycine yielded only moderate activity the other 3 amino compounds

reacted with vitamin K 1 to produce mutagens at significant levels.

CONCLUSIONS

Glycine yielded moderate mutagenic activity in Salmonella strain TA98 when tested with

vitamin K1.

STUDY RELIABILITY

[3] Not reliable

.

REFERENCE

Vithayathil et al., "Heat

- induced mutagen formation from creatine and fat

- soluble constituents of

food", 1983. Mutation Research 121: 167

- 170. FASP Study No. 85Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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43 Study 5: In vitro gene mutation in Salmonella

Title: Primary mutagenicity screening of food additives currently used in Japan

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: C rystal; 99.8% purity

METHOD

Method/guideline followed: Methods of Ames, McCann & Yamasaki (1975)

Test type: Ames

GLP compliant?: Not stated

Year study performed: 1983

Test species: Salmonella strains TA92, TA1535, TA100, TA1537, TA94 and TA98

Metabolic activation: with S9

Solvent: P hosphate buffer

Concentrations tested: U p to 35 mg/plate (highest non

- cytotoxic dose used in the

experime nt)

Statistical methods used: Not stated

Test method/conditions remarks: Cells cultured overnight were pre

- incubated with both the

test sample and the S

- 9 mix for 20 min at 37oC before plating. Duplicate plates were used for

each of six different concen trations of the sample. The number of revertant (his + ) colonies was

scored after incubation at 37°C for 2 days. The result was considered positive if the number of

colonies found was twice the number in the control (exposed to the appropriate solvent or

untreated). If no reasonable dose response was detected, additional experiments using different

doses or induced mutation frequency assays were performed.

RESULTS

Genotoxicity result: NegativeDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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44 Results remarks: No significant increases in the numbers of revertant colonies were detected in

any S. typhimurium strains at the maximum dose tested.

CONCLUSIONS

Glycine was not mutagenic in this assay.

STUDY RELIABILITY

[1] Reliable without restrictions

.

REFERENCE

Ishidate et al., "Primary Mutagenicity Screening of Food Additives Currently Used in Japan".

Fd. Chem. Toxic. Vol. 22, No. 8. pp. 623

- 636, 1984. FASP Study No. 86 & 86.1Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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45 Study 6: In vitro gene mutation in Salmonella

Title: Development and validation of the spiral Salmonella assay: an aut omated approach to

bacterial mutagenicity testing

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: F rom BioRad

METHOD

Method/guideline followed: Standard Plate

- I ncorporation Assay and Spiral Salmonella Assay

Test type: Reverse mutation

GLP compliant?: Not stated

Year study performed: 1988

Test species: Salmonella strains TA98 and TA100

Metabolic activation: W ith and without S9

Solvent: S terile water

Concent rations tested: 0.373

– 7.345μg/plate

Statistical methods used: S ee test methods remarks section

Test method/conditions remarks: The performance of the spiral Salmonella assay was

compared to that of the conventional plate

- incorporation assay using 13 mu tagens and 7

nonmutagens selected from a variety of I chemical classes.

Standard Plate

- Incorporation Assay

The plate

- incorporation assay was performed as recommended by Maron and Ames (1983)

except that minimal histidine and biotin were added to the plati ng medium rather than to the soft

agar overlay. Doses were selected so as to match the data points specified along the spiral assay

dose

- response curve (i.e., the average dose

- equivalent administered per revolution of the spiral).

Duplicate plates were p oured at each dose. 2

- Aminoanthracene (0.5 μg/plate with S9 for strains

TA98 and TA100; 3 μg/plate with S9 for strain TA1537), 2

- nitrofluorene (3 μg/plate without S9

for strain TA98), sodium azide (3 μg/plate without S9 for strain TA100), and 9

- aminoacrid ine

(100 μg/plate without S9 for strain TA1537) served as positive controls to verify the reversionDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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46 properties of the tester strains and to monitor the activity of the exogenous activation system.

Dimethyl sulfoxide or water was used as the negative contr ol. Plates were inverted and

incubated for 48 h at 37oC. Replicate experiments were performed to demonstrate

reproducibility. Revertant colonies were counted with the vidicon colony counter as well as with

the laser scanner in the pour

- plate counting mo de with no count corrections other than colonies

added for the excluded area of the agar meniscus. (Although corrected counts can be generated

for standard pour plates counted on the laser scanner using the computer program provided by

Spiral System Instr uments, Inc.

Spiral Salmonella Assay

Prior to its use, the sample delivery system of the spiral plater was sterilized by aspirating

ethanol (70%) into the stylus tip and through the Teflon tubing. The tubing was then flushed with

sterile water to remove traces of ethanol. Between applications of different bacterial strains, the

stylus assembly was flushed with ethanol and sterile water to prevent cross

- contamination.

Between applications of different chemical compounds, the

tubing was flushed repeatedly with DMSO.

The concentrated tester strain was delivered onto a rotating pre

- poured agar plate using the

uniform cam. When the liquid had absorbed into the agar and the deposition pattern was no

longer visible (< 2 min), solutions of the test compounds or controls were deposited onto the

same spiral track using the variable cam, generating a chemical concentration gradient along the

bacterial spiral. The S9 mix

- when required

– was dispensed along the same spiral path using

the variable cam. This techniq ue allowed a uniform density of bacteria to be exposed to

continuously decreasing dilutions of the chemical compound and a gradient of S9.

Duplicate plates were prepared for each compound and control tested, and experiments were

replicated to demonstrate assay reproducibility. Prepared plates were inverted and incubated for

48 h at 37 o C. Fig. 3 illustrates a typical mutagenic response on a spiral plate.

Revertant colonies were counted using the laser scanner in the variable

- dilution count mode and

a cou nt

- correction algorithm based on crowding and colony size was utilized as discussed

previously. Dose

- response data were generated for each spiral plate using the data analysis

program provided by Spiral System Instruments, Inc., and corresponding data poi nts from

duplicate plates were averaged to produce a single plot. Spontaneous control values were

obtained by averaging the 13 data points generated by each control plate, and then averaging

replicate control plates to produce a single spontaneous value.

RESULTS

Genotoxicity result: Negative

Results remarks: In general, toxicity was expressed at a lower dose in the spiral assay, and the

mutagenic potencies were greater in the spiral assay relative to the plate

- incorporation assay.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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47 CONCLUSIONS

Glyc ine was not mutagenic in this assay

.

STUDY RELIABILITY

[2] Reliable with restrictions

- The primary purpose of this research was to determine the validity

of test results generated by an automated approach to bacterial mutagenicity testing by

comparing r esponses obtained using the automated method to responses obtained using the

conventional plate

- incorporation assay.

REFERENCE

Houk et al., "Development and validation of the spiral Salmonella assay: An automated approach

to bacterial mutagenicity testi ng". Mutation Research 223: 49

- 64, 1989. FASP Study No. 91Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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48 B

- 8. CHROMOSOMAL ABERRATIONS STUDY

Summary of endpoint: Adequate

Study 1: Chromosomal aberration study

Title: Primary mutagenicity screening of food additives currently used in Japan

T he study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: C rystal, 99.8% purity

METHOD

Method/guideline followed: Not stated

Test type: I n vitro chromosomal aberration assay

GLP compliant?: Not stated

Year study performed: 1983

Test species: Chinese hamster fibroblast cell line

Metabolic activation: Not stated

Solvent: P hysiological saline

Concentrations tested: U p to 1.0 mg/mL (highest non

- cytotoxic dose used in the ex periment)

Statistical methods used: Not stated

Test method/conditions remarks: Chromosomal aberration tests were carried out on 242 food

additives using a Chinese hamster fibroblast cell line; (CHL). The cells were exposed to each

sample at three dif ferent doses for 24 and 48 h ours. In the present studies, no metabolic

activation systems were applied. The maximum dose of each sample was selected by a

preliminary test in which the dose needed for 50% cell

- growth inhibition was estimated using a

c ell densitometer.

Chromosome preparations were made as follows: Colcemid (final concentration 0.2 μg/ml) was

added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended

in a hypotonic KC1 solution (0.075 M) for 13 min at room temperature. After centrifugation theDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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49 cells were fixed with acetic acid

- methanol (l:3, v/v) and spread on clean glass slides. After air

drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12

- 1 5 min.

A hund red well

- spread metaphases were observed under the microscope (x 600 with a no

- cover

objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal

aberrations such as chromatid or chromosome gaps, breaks, exchanges, r ing formations,

fragmentations, and others, was recorded on each culture plated. Untreated cells and solvent

treated cells served as negative controls, in which the incidence of aberrations was usually less

than 3.0%. The results were considered to be n egative if the incidence was less that 4.9%,

equivocal if it was between 5.0 and 9.9% and positive it was more than 10.0%. When no

reasonable dose

- response relationships were found, additional experiments were carried out at

similar dose levels.

RESULT S

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): > 1.0 mg/mL

Results remarks: Incidence of polyploid cells at 48 hours after treatment: 1.0%; Incidence of

cells with structural chromosomal aberrations at 48 hours after treatment: 1%.

CONCLUSIONS

Glycine did not induce chromosomal aberrations in this assay

STUDY RELIABILITY

[1] Reliable without restrictions

.

REFERENCE

Ishidate et al., "Primary Mutagenicity Screening of Food Additives Currently Used in Japan".

Fd. Chem. Toxic . Vol. 22, No. 8. pp. 623

- 636, 1984. FASP Study No. 86 & 86.1Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

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50 Study 2: Sister chromatid exchange study

Title: Genetic Toxicity in vitro

The study is: Adequate Not Adequate

This study was considered inadequate du e to missing critical study information.

TEST SUBSTANCE

Identity: Glycine, CAS No. 56

- 40

- 6

Remarks: None stated

METHOD

Method/guideline followed: Not stated

Test type: Sister chromatid exchange assay

GLP compliant?: Not stated

Year study pe rformed: Not stated

Test species: H uman lymphocytes

Metabolic activation: Not stated

Concentrations tested: 100 mg/L

Statistical methods used: Not stated

Test method/conditions remarks: Not stated

RESULTS

Genotoxicity result: Positive

Cytotoxic co ncentration (if applicable): Not stated

Results remarks: Not stated

CONCLUSIONS

Glycine does initiate sister chromatid exchange in human lymphocytes at a concentration of 100

mg/L.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

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51 STUDY RELIABILITY

[4] Not assignable

– no study detail provided

.

REF ERENCE

European Commission, European Chemicals Bureau. IUCLID Dataset. 18

- Feb

- 2000.

Mutation Research (Elsevier Science Pub. B.V., POB 211, 1000 AE Amsterdam, Netherlands)

V.1

- 1964

- 280, 279, 92.Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

52 B

- 9. SKIN IRRITATION

Study 1: Dermal irritation st udy in rabbits

Title: Disodium Iminodiacetic Acid (DSIDA): Skin Irritation (Albino Rabbits)

TEST SUBSTANCE

Identity: Disodium imino diacetic acid, CAS No. 928

- 72

- 3

Remarks: ID No. 3

- 18

- 85

- 1500, undiluted, warmed to 50 deg C

METHOD

Method/Guide line followed: Not stated

Test Type: Skin irritation

GLP: Yes

Year study performed: 1985

Species/Strain: New Zealand white rabbits

Sex: Both

Number of animals per dose: 6

Age and body weight range of animals used: Not specified

Route of admi nistration: Dermal

Vehicle: None

Dosing regimen : Application of the test substance was made to intact skin in both the front and

rear of the animal under semi

- occlusive dressings for an exposure period of 4 hours.

Observations were made for erythema a nd edema after 1, 24, 48 and 72 hours following patch

removal.

Doses: 0.5 mL

Statistical methods used: Not stated

Remarks: NoneDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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53 RESULTS

Mean scores for erythema and edema were 2.0 at up to 72 hours, and 0.0 at 168 hours. Skin was

observed to peel off in seven to ten days. There was no injury in depth.

CONCLUSIONS

No conclusion noted.

RELIABILITY CODE

[ 2 ] Reliable with restrictions.

REFERENCE

Disodium Iminodiacetic Acid (DSIDA): Skin Irritation (Albino Rabbits). Study Number: YO

85

- 073

. Obta ined from 8d 8607

- 0000045 Monsanto.pdf: page 1252. Received November 20,

2006

Disk 3, p. 5269, 5273Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

54 Study 2: Dermal irritation study in rabbits

Title: Disodium Iminodiacetic Acid (DSIDA): Skin Irritation (Albino Rabbits)

TEST SUBSTANCE

Identity: Disodium imino diacetic acid, CAS No. 928

- 72

- 3

Remarks: ID No. 3

- 18

- 85

- 1500, undiluted, warmed to 50 deg C

METHOD

Method/Guideline followed: Not stated

Test Type: Skin irritation

GLP: Yes

Year study performed: 1985

Species/Strain: New Zealand W hite rabbits

Sex: Both

Number of animals per dose: 6

Age and body weight range of animals used: Not specified

Route of administration: Dermal

Vehicle: None

Dosing regimen : Application of the test substance was made on intact skin of both the front and

rear of the animal under occlusive dressings for a 24

- hour exposure period. Observations of

erythema and edema were made 24, 48, 72 and 166 hours following removal of the patch

application.

Doses: 0.5 mL

Statistical methods used: No t stated

Remarks: NoneDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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55 RESULTS

Mean scores were 8.0 for erythema and edema at 24, 48, 72, and 166 hours. The test substance

was determined to be corrosive. Observations included loosening about the edges of scab in ten

to fourteen days showing injury in depth.

CONCLUSIONS

The substance was considered corrosive.

RELIABILITY CODE

[ 2 ] Reliable with restrictions.

REFERENCE

Disodium Iminodiacetic Acid (DSIDA): Skin Irritation (Albino Rabbits). Study Number: YO

85

- 073

. Obtained from 8d 8607

- 0000045 Mo nsanto.pdf: page 1253. Received November 20,

2006

Disk 3, p. 5269

, 5274Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

56 B

- 10. EYE IRRITATION

Study 1: Eye irritation study in rabbits

Title: Disodium Iminodiacetic Acid (DSIDA): Eye Irritation (Albino Rabbits)

TEST SUBSTANCE

Identity: Disodi um imino diacetic acid, CAS No. 928

- 72

- 3

Remarks: ID No. 3

- 18

- 85

- 1500; undiluted; warmed to 50 deg C

METHOD

Method/Guideline followed: Not stated

Test Type: Eye irritation

GLP: Yes

Year study performed: 1985

Species/Strain: New Zealand white ma le and female rabbits

Sex: Both

Number of animals per dose: 6

Age and body weight range of animals used: Not stated

Route of administration: Ocular

Vehicle: None

Dosing regimen : The substance was placed in the conjunctival sac of one eye of each rabbit.

Exposure lasted 24 hours.

Doses: 0.1 mL

Statistical methods used: Not stated

Remarks: None statedDraft HPV4 Chemical Data Adequacy Review CAS No. 928

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57 RESULTS

Immediate observations included severe discomfort with pawing, squealing, thrashing about

stocks and eyes tightly closed.

CONCLUSIONS

The test substance was noted as corrosive in under a minute following exposure.

RELIABILITY CODE

[ 2 ] Reliable with restrictions.

REFERENCE

Disodium Iminodiacetic Acid (DSIDA): Eye Irritation (Albino Rabbits). Study Number: YO

- 85

-

073

. Obtained from 8 d 8607

- 0000045 Monsanto.pdf: page 1251. Received November 20, 2006

Disk 3, p. 5269

, 5272Draft HPV4 Chemical Data Adequacy Review CAS No. 928

- 72

- 3

58 CAS No. 928

- 72

- 3: Summary of Endpoints

Endpoint Study Information

Provided Acceptable Data Gap 1

Aquatic Effects

Acute Toxicity to Fish Yes Yes No

Acute Toxicity to Daphnia Yes Yes No

Toxicity to Aquatic Plants Yes Yes No

Health Effects

Acute Toxicity Yes Yes No

Repeated

- Dose Toxicity Yes No No

Genetic Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Toxicity to Reproduction No No No

Developmental To xicity No No No

1

1 The analog, glycine, has had no toxic effects associated with dietary exposures or use as a

dietary supplement , although some people have reported that taking this supplement causes

stomach upset. Therefore , the aquatic and heal th effects endpoints with inadequate or no

available data were not considered to be data gaps. See the “Supporting Information” section of

the glycine DA/RS document (2010) for excerpts from public literature which supports this

claim . Although no experi mental data exist on aquatic toxicity of glycine, based on the above

information, the aquatic toxicity of glycine is expected to be low.