SRC TR-10-114

HPV4 CHEMICAL DATA ADEQUACY 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

Chemical, 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, 2010
HPV4 Chemical Data Adequacy Review

Glycine, N-(carboxymethyl)-, sodium salt, CAS No. 928-72-3

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 22202
Draft 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, inhalation) 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 - Bibliographic database that contains references to journal articles in the life 

sciences with a concentration on biomedicine

TOXNET -National Library of Medicine 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 System (CCRIS) -

Carcinogenicity and mutagenicity test results for over 8000 

• chemicals);

• Hazardous Substances Data Bank (HSDB) - Comprehensive, peerreviewed toxicology data for about 5000 chemicals;

• Genetic Toxicology Data Bank (GENE-TOX) - Peer-reviewed genetic 

toxicology test data for over 3000 chemicals.

TSCATS - Unpublished 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 database
Draft 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 Chemical Safety Concise International Chemical Assessment 

Documents (IPCS INCHEM or CICADs)

European Chemical Substances Information System (ESIS) - source of IUCLID Datasets

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

submitted 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 Datasets sometimes contained studies of interest, certain study 

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

of the study, if available and accessible, was obtained and reviewed.

A-2. Evaluating data adequacy

Using standard OECD testing guidelines and professional 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 standard 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 if 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 page 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 summary 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 ECOSAR version 1.00 

Molecular weight = 177.07

Log Kow = -7.51

Water solubility = 1E+006 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 = 1.64e+007 mg/L

Results remarks: 

96-hour LC50 was estimated from ECOSAR version 1.00
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

4

CONCLUSIONS:

The predicted 96h LC50 for fish is 1.64e+007 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions – endpoint value estimated using a modeling program.

REFERENCE

ECOSAR version 1.00
Draft 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 = -7.51

Water solubility = 1E+006 mg/L

Test type: Acute 48-hour toxicity estimate

GLP compliant?: N/A

Year study was performed: N/A

Test species: Daphnids

Analytical monitoring: N/A

Exposure duration: 48 hours

Concentrations tested: N/A

Test method/conditions remarks: N/A

RESULTS

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

Results remarks:

48-hour LC50 was estimated from ECOSAR version 1.00
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

6

CONCLUSIONS

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

STUDY RELIABILITY

[2] Reliable with restrictions – endpoint value estimated using a modeling program.

REFERENCE

ECOSAR version 1.00
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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

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 = -7.51

Water solubility = 1E+006 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/conditions remarks: N/A

RESULTS

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

Results remarks:

96-hour EC50 was estimated from ECOSAR version 1.00
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

8

CONCLUSIONS

The predicted 96h EC50 for green algae is 4527.094 mg/L.

STUDY RELIABILITY

[2] Reliable with restrictions – endpoint value estimated using a modeling program.

REFERENCE

ECOSAR version 1.00
Draft 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 glycine, N-(carboxymethyl)-, sodium salt.

Study 1: Acute oral toxicity study in rats

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

The study is: 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 toxicity

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 administration: 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 methods used: Not stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 females 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 gastrointestinal 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 restrictions.

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. 5269-5270
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

11

Study 2: Acute dermal toxicity 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 made 

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. 928-72-3

12

CONCLUSIONS

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: page 1250. Received 

November 20, 2006

Disk 3, p. 5269, 5271
Draft 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 missing 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 species: 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

Endpoint value(s): 7930 mg/kg bw

Results remarks: No data
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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. 90
Draft 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.

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: Mouse, 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

Endpoint value(s): 4920 mg/kg bw

Results remarks: No data
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 Chiryo. Pharmacology and Theraputics (Raifu Saiensu Shuppan K. K., 2-5-

13, Yaesu, Chuo-ku, Tokyo 104, Japan. V.1 – 1972-5, 1502, 77
Draft 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 followed: 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 data

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 5200 mg/kg bw

Results remarks: No data
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

18

CONCLUSIONS

The acute s.c. LD50 in rats is 5200 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, 77

Lewis, R.J., Food Additives Handbook p. 235-236 (1989); FASP Study No. 90
Draft 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 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.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 data
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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, Yaesu, Chuo-ku, Tokyo 104, Japan. V.1 – 1972-5, 1502, 77
Draft 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 performed: 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 data
Draft 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.

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
Draft 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 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): 2600 mg/kg-bw

Results remarks: No data
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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, 77
Draft 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 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 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 data
Draft 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 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, 77
Draft 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 considered 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 data

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

Statistical methods used: No data

Test method/conditions remarks: No data

RESULTS

Endpoint value(s): 3 g/kg

Results remarks: No data
Draft 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, 306, 42
Draft 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 reported. Additionally, limited study details were provided.

TEST SUBSTANCE 

Identity: Glycine, CAS No. 56-40-6

Remarks: Purity not reported

METHOD

Method/guideline followed: None stated

Test type: Short term dietary study

GLP compliant?: Not stated

Year study performed: 1949

Test species: Sprague-Dawley weanling rats

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

Route of administration: Oral, diet

Vehicle: Food 

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 37th and 46th 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 approximately 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 content of 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 food 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 ascorbic 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. 928-72-3

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. Hemoglobin 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 various 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 hemoglobin 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 growthpromoting effect of PGA when added to a high glycine diet 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 improvement 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 control 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 Acid, 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. 928-72-3

32

B-6: CARCINOGENICITY

Study 1: Chronic toxicity/carcinogenicity 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 

METHOD

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

Test 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: Drinking 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 the Wilcoxon t-test or the x2

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) received 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 samples being then taken from all animals of both sexes in each group for 
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 autopsy 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 

stained 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 diet 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 group, 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 urinalysis 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 

significantly 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 weights. 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 females 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 other 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 calcification 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. 928-72-3

34

CONCLUSIONS

Glycine was carcinogenic to rats in this study.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

Kitahori et al., 1994. Carcinogenicity Study 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. 928-72-3

35

B-7: GENE MUTATION STUDIES

Summary of endpoint: Adequate

Study 1. 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, 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, 3333 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 TA1537 at a concentration of 3333 μg/plate only.
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

36

Results remarks: Not stated

CONCLUSIONS

Glycine was not mutagenic in this assay.

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE

NTP (1979). Salmonella (152901). 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. 928-72-3

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, 100, 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 metabolic 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 assay.
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

38

STUDY RELIABILITY

[1] Reliable without restrictions.

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. 928-72-3

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: Adequate 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 study 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: Plate assay for mutagenesis: Logarithmic-phase cells were 

prepared by diluting overnight cultures 1:50 into warm SMM1, and shaking the cultures at 37°C 

until a concentration of 4 x 108 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 108

CFU/ml. Cells (0.1 ml) were spread on Glu-600 plates (lactose plates containing a growthlimiting 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 scoring 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 on 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 

preexisting mutants per plate were 1(wild-type), 15(uvrB), and 0(uvrB umuC and uvrB lexA), 
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

40

while the mean number of plate mutants per control plate were 60(wild-type), 355(uvrB), 

21(uvrB umuC), and 20(uvrB lexA). For Glu-600 control plates, the total Laccells per plate was

3.4 X 1010 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.

REFERENCE

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. 83
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

41

Study 4: In vitro gene mutation in Salmonella

Title: Mutagenicity of heated solutions of Vitamin K1 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 K1, 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: Gene mutation in Salmonella

GLP compliant?: Not stated

Year study performed: 1983

Test species: Salmonella strain TA98

Metabolic activation: With S9

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

mutagenicity.

Statistical methods used: Not stated

Test method/conditions remarks: Since vitamin K1 reacted with creatine to yield exceptionally 

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

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 100 
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 were 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 rat-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 K1 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. 85
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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: Crystal; 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: Phosphate buffer

Concentrations tested: Up to 35 mg/plate (highest non-cytotoxic dose used in the 

experiment)

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 concentrations 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: Negative
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 & 86.1
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

45

Study 6: In vitro gene mutation in Salmonella

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

bacterial mutagenicity testing

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Glycine, CAS No. 56-40-6

Remarks: From BioRad

METHOD

Method/guideline followed: Standard Plate-Incorporation 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: With and without S9

Solvent: Sterile water

Concentrations tested: 0.373 – 7.345μg/plate

Statistical methods used: See 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 mutagens 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 plating 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 poured 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-aminoacridine 

(100 μg/plate without S9 for strain TA1537) served as positive controls to verify the reversion 
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 control. 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 mode 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 Instruments, 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 technique 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 37oC. 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 count-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 points 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. 928-72-3

47

CONCLUSIONS

Glycine 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 responses 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 testing". Mutation Research 223: 49-64, 1989. FASP Study No. 91
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Glycine, CAS No. 56-40-6

Remarks: Crystal, 99.8% purity

METHOD

Method/guideline followed: Not stated

Test type: In vitro chromosomal aberration assay

GLP compliant?: Not stated

Year study performed: 1983

Test species: Chinese hamster fibroblast cell line

Metabolic activation: Not stated

Solvent: Physiological saline

Concentrations tested: Up to 1.0 mg/mL (highest non-cytotoxic dose used in the experiment)

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 different 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 

cell 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 the 
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

49

cells were fixed with acetic acid-methanol (l:3, v/v) and spread on clean glass slides. After airdrying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-1 5 min. 

A hundred 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, ring formations, 

fragmentations, and others, was recorded on each culture plated. Untreated cells and solventtreated cells served as negative controls, in which the incidence of aberrations was usually less 

than 3.0%. The results were considered to be negative 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. 

RESULTS

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 & 86.1
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

50

Study 2: Sister chromatid exchange study

Title: Genetic Toxicity in vitro

The study is: Adequate Not Adequate

This study was considered inadequate due 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 performed: Not stated

Test species: Human 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 concentration (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. 928-72-3

51

STUDY RELIABILITY

[4] Not assignable – no study detail provided.

REFERENCE

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 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 white 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 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 and edema after 1, 24, 48 and 72 hours following patch 

removal.

Doses: 0.5 mL

Statistical methods used: Not stated

Remarks: None
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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: YO85-073. Obtained from 8d 8607-0000045 Monsanto.pdf: page 1252. Received November 20, 

2006

Disk 3, p. 5269, 5273
Draft 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 White 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: Not stated

Remarks: None
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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: YO85-073. Obtained from 8d 8607-0000045 Monsanto.pdf: page 1253. Received November 20, 

2006

Disk 3, p. 5269, 5274
Draft 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: 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: Eye irritation

GLP: Yes

Year study performed: 1985

Species/Strain: New Zealand white male 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 stated
Draft HPV4 Chemical Data Adequacy Review CAS No. 928-72-3

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 8d 8607-0000045 Monsanto.pdf: page 1251. Received November 20, 2006

Disk 3, p. 5269, 5272
Draft 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 Gap1

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 Toxicity 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 health 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 experimental data exist on aquatic toxicity of glycine, based on the above 

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