SRC TR-10-024

HPV4 CHEMICAL DATA ADEQUACY REVIEW

1H-Benzotriazole, 4(or 5)-methyl-, CAS No. 29385-43-1

Prepared by:

Christopher Janson, Carlye Austin, and Joan D. Garey

Environmental Science 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, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

February 9, 2010
HPV4 Chemical Data Adequacy Review

1H-Benzotriazole, 4(or 5)-methyl-, CAS No. 29385-43-1

February 2010

DRAFT

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. 

Environmental Science Center 

2451 Crystal Drive, Suite 804 

Arlington, VA 22202
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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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
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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.
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B-1. ACUTE TOXICITY TO FISH

Summary of endpoint: Adequate 

Study 1: Acute toxicity study in fathead minnows 

Title: Comparative Measures of the Toxicity of Component Chemicals in Aircraft Deicing Fluid

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 5-Methyl-1H-Benzotriazole or 4-Methyl-1H-Benzotriazole (MeBT), CAS No. 29385-

43-1

Remarks: Provided by Cincinnati Specialties (Cincinnati Ohio)

METHOD

Method/guideline followed: U.S. Environmental Protection Agency. 1985. Methods for 

measuring the acute toxicity of effluents and receiving waters to freshwater and marine 

microorganisms. EPA 600/4-85-013. Environmental Monitoring and Support Laboratory, 

Cincinnati, OH.

Test type: Acute LC50; static-renewal renewed at 50 hours with freshly-prepared dilutions

GLP compliant?: Not stated

Year study was performed: ~1999

Test species: Fathead minnows (Pimephales promelas). Juvenile fathead minnows used in this 

study were obtained from in-house cultures, and less than 7 days old at test initiation.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 150.8 and 188.9 mg/L

Test method/conditions remarks: The toxicity of MeBT to fathead minnows was assessed in 

moderately hard water using acute tests according to U.S. Environmental Protection Agency 

procedures. Mortality was the endpoint. The nominal (predilution) test concentrations of MeBT 

were 150.8 and 188.9 mg/L. A dilution water control was included. Test chambers were 500-

mL glass beakers. All tests were conducted at 20 oC with a light:dark cycle of 16:8 hours. 

Fathead minnows were fed 0.2 mL of brine shrimp nauplii (Artemia sp.) at 48 hrs and were 

renewed two hours after feeding with freshly prepared dilutions. LC50 values were calculated 
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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using either the graphical, probit, Spearman-Karber, or trimmed Spearman-Karber methods 

according to U.S. Environmental Protection Agency established criteria.

RESULTS

Endpoint value(s): The 96-hour LC50 value for fathead minnows was 38-65 mg/L.

Results remarks: Measured high concentrations of MeBT were between 95.5 and 99.5% of 

nominal concentrations at a confidence interval of 95%. Lower test concentrations were 

calculated as a proportion of the 100% measured concentration. The LC50 was calculated based 

on the measured 100% concentration.

CONCLUSIONS

The data provided by this study confirmed that MeBT contributes to the toxicological properties 

of aircraft deicing fluid formulations.

STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

Cornell, J.S. et al. 2000. Comparative measures of the toxicity of component chemicals in 

aircraft deicing fluid. Environmental Chemistry, 19(6): 1465-1472.
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Study 2: Acute toxicity study in fathead minnows 

Title: The Acute Toxicity of Tolyltriazole-Cobratec TT-100 Sample 1090 to the Fathead 

Minnow Pimephales promelas

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Tolyltriazole-Cobratec TT-100, Sample 1090. Supplied as tan 

granules.

METHOD

Method/guideline followed: Based on that suggested in “Fish – Pesticide Acute Toxicity Test 

guideline” issued by the Environmental Protection Agency; Pesticides Regulation Division; 

January 20, 1971.

Test type: Acute LC50

GLP compliant?: Not specified

Year study was performed: 1974

Test species: Pimephales promelas. Obtained from Kurtz’s Fish Hatchery; Elverson, 

Pennsylvania.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 17.80, 22.40, 28.20, 35.50 ppm

Test method/conditions remarks: Average length of fish was 65 mm; average weight of fish 

was 1.2 g.

Fish were held in a plastic wading pool prior to bioassay. The water temperature of the pool was 

68 ±2 oF. Fish were held for 10 days before bioassay.

Test Medium –

Container: 5 gallon glass water bucket lined with a clear polyethylene bag which was discarded 

and replaced after each use.

Water Quantity: 12 liters per test vessel.

Water Quality: Standard reconstituted distilled water.
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Water Temperature: 68 ±2 oF.

Test Chemicals –

Candidate: Tolyltriazole-Cobratec TT-100, Sample 1090, (technical grade chemical) supplied by 

Sherwin-Williams Chemicals.

Solvent, acetone (0.3 mL/liter of test water)

Reference: p, p’DDT, 74%

Solvent, acetone (0.3 mL/liter of test water)

Determinations of Lethal Concentrations –

Screening tests using 2 fish per dose level were conducted to locate the general level of toxicity 

of Tolyltriazole-Cobratec TT-100, Sample 1090, and 74% p, p’DDT to the particular lot of fish.

From the results of the screening tests, the following concentrations were chosen as the doses to 

use in the bioassay:

Cobratec TT-100 (ppm): 17.80, 22.40, 28.20, 35.50

74% p, p’DDT (ppm): 0.0563, 0.0709, 0.0892, 0.1120, 0.1410

The test lasted for 96 hours with mortality readings taken every 24 hours.

Twenty fish were exposed to each of the above concentrations. The fish were loaded five to a 

test vessel (0.5 gm fish/liter of water). Twenty fish were untreated and served as controls.

Reaction of fish: 

1. Controls – there were no deaths and no signs of intoxication.

2. Tolyltriazole – Cobratec TT-100, Sample 1090 – the major symptom of toxicity was loss of 

equilibrium prior to death.

3. 74%, p, p’DDT – death was usually preceded by loss of equilibrium.

Litchfield and Wilcoxon’s method was used to compute the confidence limits for the LC1, LC50, 

and LC99 (at 24, 48 and 96 hours).

RESULTS

Endpoint value(s):

24 hour:

LC1 = 23.00 ppm

LC50 = 36.90 ppm

LC99 = 68.50 ppm

48 hour:

LC1 = 14.00 ppm

LC50 = 29.80 ppm

LC99 = 62.50 ppm
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96 hour:

LC1 = 14.20 ppm

LC50 = 25.50 ppm

LC99 = 46.00 ppm

Results remarks: 

The positive control (74% p, p’DDT) data are as follows: 

24 hour:

LC1 = 0.0460 ppm

LC50 = 0.1180 ppm

LC99 = 0.3010 ppm

48 hour:

LC1 = 0.0485 ppm

LC50 = 0.1010 ppm

LC99 = 0.2110 ppm

96 hour:

LC1 = 0.0360 ppm

LC50 = 0.0945 ppm

LC99 = 0.2980 ppm

CONCLUSIONS

It is concluded that Cobratec TT-100 is less toxic to the fathead minnow than is 74% p, p’DDT.

STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

TSCATS submission (OTS0521120, Doc ID 86-890000612; received 11/06/74): Huntingdon 

Research Center, Sherwin Williams Company. 1974. Initial submission: The Acute Toxicity of 

Tolyltriazole-Cobratec TT-100 Sample 1090 to the Fathead Minnow (Pimephales promelas) with 

Attachments and Cover Letter dated 061289.
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Study 3: Acute toxicity study in bluegill sunfish 

Title: The Acute Toxicity of Tolyltriazole-Cobratec TT-100 Sample 1090 to the Bluegill Sunfish 

(Lepomis macrochirus)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Tolyltriazole-Cobratec TT-100, Sample 1090. Supplied as tan 

granules.

METHOD

Method/guideline followed: Based on that suggested in “Fish – Pesticide Acute Toxicity Test 

guideline” issued by the Environmental Protection Agency; Pesticides Regulation Division; 

January 20, 1971.

Test type: Acute LC50

GLP compliant?: Not indicated

Year study was performed: 1974

Test species: Bluegill sunfish (Lepomis macrochirus). Obtained from Kurtz’s Fish Hatchery; 

Elverson, Pennsylvania.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 3.16, 3.55, 3.98, 4.47 ppm

Test method/conditions remarks: Average length of fish was 42 mm; average weight of fish 

was 0.8 g.

Fish were held in a plastic wading pool prior to bioassay. The water temperature of the pool was 

72 ±2 oF. Fish were held for 10 days before bioassay.

Test Medium –

Container: 5 gallon glass water bucket lined with a clear polyethylene bag which was discarded 

and replaced after each use.

Water Quantity: 16 liters per test vessel.

Water Quality: Standard reconstituted distilled water.
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Water Temperature: 72 ±2 oF.

Test Chemicals –

Candidate: Tolyltriazole-Cobratec TT-100, Sample 1090, (technical grade chemical) supplied by 

Sherwin-Williams Chemicals.

Solvent, acetone (0.3 mL/liter of test water)

Reference: p, p’DDT, 74%

Solvent, acetone (0.3 mL/liter of test water)

Determinations of Lethal Concentrations –

Screening tests using 2 fish per dose level were conducted to locate the general level of toxicity 

of Tolyltriazole-Cobratec TT-100, Sample 1090, and 74% p, p’DDT to the particular lot of fish.

From the results of the screening tests, the following concentrations were chosen as the doses to 

use in the bioassay:

Cobratec TT-100 (ppm): 3.16, 3.55, 3.98, 4.47

74% p, p’DDT (ppm): 0.016, 0.013, 0.010, 0.008

The test lasted for 96 hours with mortality readings taken every 24 hours.

Twenty fish were exposed to each of the above concentrations. The fish were loaded ten to a test 

vessel (0.5 gm fish/liter of water). Twenty fish were untreated and served as controls.

Reaction of fish: 

1. Controls – there were no deaths and no signs of intoxication.

2. Tolyltriazole – Cobratec TT-100, Sample 1090 – the major symptom of toxicity was loss of 

equilibrium prior to death.

3. 74%, p, p’DDT – death was usually preceded by loss of equilibrium.

Litchfield and Wilcoxon’s method was used to compute the confidence limits for the LC1, LC50, 

and LC99 (at 24, 48 and 96 hours).

RESULTS

Endpoint value(s):

24 hour:

LC1 = 3.90 ppm

LC50 = 4.02 ppm

LC99 = 4.81 ppm

48 hour:

LC1 = 2.55 ppm

LC50 = 3.55 ppm

LC99 = 4.90 ppm
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96 hour:

LC1 = 2.30 ppm

LC50 = 3.47 ppm

LC99 = 5.20 ppm

Results remarks: 

The positive control (74% p, p’DDT) data are as follows: 

24 hour:

LC1 = 0.0084 ppm

LC50 = 0.0126 ppm

LC99 = 0.0192 ppm

48 hour:

LC1 = 0.0063 ppm

LC50 = 0.0110 ppm

LC99 = 0.0192 ppm

96 hour:

LC1 = 0.0065 ppm

LC50 = 0.0106 ppm

LC99 = 0.0172 ppm

CONCLUSIONS

It is concluded that Cobratec TT-100 is less toxic to the bluegill sunfish than is 74% p, p’DDT.

STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

TSCATS submission (OTS0521121, Doc ID 86-890000613, received 11/06/74): Huntingdon 

Research Center, Sherwin Williams Company. 1974. Initial submission: The Acute Toxicity of 

Tolyltriazole-Cobratec TT-100 Sample 1090 to the Bluegill Sunfish (Lepomis macrochirus) with 

Attachments and Cover Letter Dated 061289.
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Study 4: Acute toxicity study in bluegill sunfish 

Title: The Acute Toxicity of Tolyltriazole-Cobratec TT-100 Sample 1090 to the Bluegill Sunfish 

(Lepomis macrochirus)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Tolyltriazole-Cobratec TT-100, Sample 1090. Supplied as tan 

granules.

METHOD

Method/guideline followed: Based on that suggested in “Fish – Pesticide Acute Toxicity Test 

guideline” issued by the Environmental Protection Agency; Pesticides Regulation Division; 

January 20, 1971.

Test type: Acute LC50

GLP compliant?: Not indicated

Year study was performed: 1975

Test species: Bluegill sunfish (Lepomis macrochirus). Obtained from Kurtz’s Fish Hatchery; 

Elverson, Pennsylvania.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 25.1, 28.2, 31.6, 35.5, 39.8 and 44.7 ppm

Test method/conditions remarks: Average length of fish was 38 mm; average weight of fish 

was 0.9 gm.

Fish were held in a plastic wading pool prior to bioassay. The water temperature of the pool was 

72 ±2 oF. Fish were held for 10 days before bioassay.

Test Medium –

Container: 5 gallon glass water bucket

Water Quantity: 18 liters per test vessel.

Water Quality: Standard reconstituted distilled water.

Water Temperature: 72 ±2 oF.
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Test Chemicals –

Candidate: Tolyltriazole-Cobratec TT-100, Sample 1090, (technical grade chemical) supplied by 

Sherwin-Williams Chemicals.

Solvent, acetone < 0.3 mL/liter of test water

Determinations of Lethal Concentrations –

Screening tests using 2 fish per dose level were conducted to locate the general level of toxicity

of Tolyltriazole-Cobratec TT-100, Sample 1090 to the particular lot of fish.

From the results of the screening tests, the following concentrations were chosen as the doses to 

use in the bioassay:

Cobratec TT-100: 25.1, 28.2, 31.6, 35.5, 39.8 and 44.7 ppm

The test lasted for 96 hours with mortality readings taken every 24 hours.

Twenty fish were exposed to each of the above concentrations. The fish were loaded ten to a test 

vessel (0.5 gm fish/liter of water). Twenty fish were untreated and served as controls.

Reaction of fish: 

1. Controls – there were no deaths and no signs of intoxication.

2. Tolyltriazole – Cobratec TT-100, Sample 1090 – the major symptom of toxicity was loss of 

equilibrium, sporadic swimming, and lying on their side prior to death

Litchfield and Wilcoxon’s method was used to compute the confidence limits at 96 hours.

RESULTS

Endpoint value(s): The LC50 is determined to be 31 PPM with 95% confidence limits of 32.3 to 

29.8 ppm.

Results remarks: 

Cobratec TT-100 (ppm) No. Dead/No. Dosed

35.5 20/20

31.6 10/20

28.2 2/20

25.1 1/20

CONCLUSIONS

None specified
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STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

TSCATS submission (OTS0521122, Doc ID 86-890000614, received 09/29/75): Huntingdon 

Research Center, Sherwin Williams Company. 1975. Initial submission: The Acute Toxicity of 

Tolyltriazole-Cobratec TT-100 Sample 1090 to the Bluegill Sunfish (Lepomis macrochirus) with 

Attachments and Cover Letter Dated 061289.
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Study 5: Acute toxicity study in rainbow trout 

Title: The Acute Toxicity of Tolyltriazole-Cobratec TT-100 Sample 1090 to the Rainbow Trout 

Salmo gairdneri

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Tolyltriazole-Cobratec TT-100, Sample 1090. Supplied as tan 

granules.

METHOD

Method/guideline followed: Based on that suggested in “Fish – Pesticide Acute Toxicity Test 

guideline” issued by the Environmental Protection Agency; Pesticides Regulation Division; 

January 20, 1971.

Test type: Acute LC50

GLP compliant?: Not indicated

Year study was performed: Not specified

Test species: Rainbow trout (Salmo gairdneri) Obtained from Kurtz’s Fish Hatchery; Elverson, 

Pennsylvania.

Analytical monitoring: Yes

Exposure duration: 96 hours

Concentrations tested: 25.1, 28.2, 31.6, 35.5, 39.8 and 44.7 ppm

Test method/conditions remarks: Average length of fish was 43 mm; average weight of fish 

was 0.35 gm.

Fish were held in an indoor tank prior to bioassay. The water temperature of the pool was 52 ±2 

F. Fish were held for 10 days before bioassay.

Test Medium –

Container: 5 gallon glass water bucket

Water Quantity: 14 liters per test vessel.

Water Quality: Standard reconstituted distilled water.

Water Temperature: 52 ±2 oF.
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Test Chemicals –

Candidate: Tolyltriazole-Cobratec TT-100, Sample 1090, (technical grade chemical) supplied by 

Sherwin-Williams Chemicals.

Solvent, acetone (0.3 mL/liter of test water)

Reference: p, p’DDT, 74%

Solvent, acetone (0/.3 mL/liter of test water)

Determinations of Lethal Concentrations –

Screening tests using 5 fish per dose level were conducted to locate the general level of toxicity 

of Tolyltriazole-Cobratec TT-100, Sample 1090, and 74% p, p’DDT to the particular lot of fish.

From the results of the screening tests, the following concentrations were chosen as the doses to 

use in the bioassay:

Cobratec TT-100 (ppm): 10.01, 14.10, 20.00, 28.20

74% p, p’DDT (ppm): 0.0200, 0.0251, 0.0316, 0.0308

The test lasted for 96 hours with mortality readings taken every 24 hours.

Twenty fish were exposed to each of the above concentrations. The fish were loaded ten to a test 

vessel (0.5 gm fish/liter of water). Twenty fish were untreated and served as controls.

Reaction of fish: 

1. Controls – there were no deaths and no signs of intoxication.

2. Tolyltriazole – Cobratec TT-100, Sample 1090 – the major symptom of toxicity was loss of 

equilibrium prior to death.

3. 74% p, p’DDT – death was usually preceded by loss of equilibrium.

Litchfield and Wilcoxon’s method was used to compute the confidence limits at 96 hours.

RESULTS

Endpoint value(s):

The 96-hour LC50 is determined to be 21.40 ppm with 95% confidence limits of 18.94 to 24.18 

ppm.

Results remarks: 

CONCLUSIONS

None specified
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STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

TSCATS submission (OTS0521123, Doc ID 86-890000615, received 06/13/89): Huntingdon 

Research Center, Sherwin Williams Company. 1974. Initial submission: The Acute Toxicity of 

Tolyltriazole-Cobratec TT-100 Sample 1090 to the Rainbow Trout (Salmo gairdneri) with 

Attachments and Cover Letter Dated 061289.
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Study 6: Acute toxicity study in fathead minnows 

Title: A 96-Hour Static-Renewal Acute Toxicity Test with the Fathead Minnow (Pimephales 

promelas)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Off-white particles

METHOD

Method/guideline followed: OECD Guideline 203

Test type: Acute LC50; static renewal

GLP compliant?: Yes

Year study was performed: 1992

Test species: Juvenile fathead minnow (Pimephales promelas). From Wildlife International Ltd. 

Mean weight: 0.33 g; standard length: 27 mm.

Analytical monitoring: Not indicated

Exposure duration: 96 hours

Concentrations tested: 0, 13, 22, 36, 60 and 100 mg/L

Test method/conditions remarks: Fathead minnows were exposed to a geometric series of five 

test concentrations and a negative (well water) control under static-renewal test conditions for 96 

hours. Two replicate test chambers were maintained in each treatment and control group, with 

10 fish in each test chamber. Nominal test concentrations were selected in consultation with the 

sponsor, and were based upon results of range finding tests. Nominal concentrations selected 

were 13, 22, 36, 60 and 100 mg/L.

Immediately prior to initiating the test, fish were impartially removed from holding tanks in 

groups of two and distributed among the test chambers until each contained 10 fish. At 48 hours, 

new test solutions were prepared and fish were transferred to the new solutions. Observations of 

mortality and other clinical signs of toxicity were made at approximately 24, 48, 72, and 96 

hours after test initiation. Cumulative percent mortality values in each treatment group were 

used to calculate LC50 values and 95% confidence limits at 24, 48, 72 and 96 hours. The no 

mortality concentration was determined by visually examining the mortality data.
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RESULTS

Endpoint value(s): The 96-hour LC50 is 43 mg/L.

Results remarks: Water temperatures taken daily with a hand-held thermometer ranged from 

21.0 to 22.9 oC. Dissolved oxygen and pH readings ranged from 8.4 to 9.2 mg/L and 7.8 to 8.5, 

respectively. Dissolved oxygen concentrations exceeded 60% of saturation throughout the test. 

Conductivity, alkalinity, and hardness measured at test initiation (0 hours) in the Negative 

Control Replicate A were 295 μmhos/cm, 188 mg/L as CaCO3, and 144 mg/L as CaCO3, 

respectively. At test termination (96 hours) they were 325 umhos/cm, 184 mg/L as CaCO3, and 

144 mg/L as CaCO3, respectively, in the Negative Control Replicate A.

After the test substance was added to the vortex, a clear colorless solution as formed in the test 

chambers. White particulate matter was noted on the bottom of one tank (Replicate A) of the 36 

mg/L treatment group, approximately 24 hours after test initiation. The particulate matter 

remained until test termination.

Fish in the negative control, with the exception of one fish which appeared lethargic at 48 hours, 

appeared healthy and normal throughout the test. The 13 and 22 mg/L groups appeared normal 

and healthy throughout the test with no signs of disease or stress. Overt signs of toxicity 

observed at the 36 mg/L concentration, beginning approximately 24 hours after test initiation, 

included lethargy, loss of equilibrium, and erratic swimming. At the 36 mg/L concentration, 

15% mortality was observed at 24 hours with no further mortality occurring by 96 hours. At the 

60 and 100 mg/L concentrations, 100% mortality was observed at 24 hours. 

CONCLUSIONS

The 96-hour LC50 value for fathead minnows exposed to test substance was 43 mg/L. The lower 

and upper 95% confidence limits were 36 and 60 mg/L. Based on visual interpretation of the 

data, the no mortality concentration was 22 mg/L.

STUDY RELIABILITY

Klimisch code = [1]. Reliable without restriction.

REFERENCE

TSCATS submission (OTS0557287, Doc ID 86940000877S, received 06/02/94): Wildlife 

International Ltd, CBI. 1993. Initial submission: A 96-hour Static Renewal Toxicity Test with 

the Fathead Minnow (Pimephales promelas) of Tolyltriazole – 1H-Benzotriazole, Methyl with 

Cover Letter Dated 04/29/94 (SANITIZED).
Draft HPV4 Chemical Data Adequacy Review CAS No. 29385-43-1

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Study 7: Acute toxicity study in bluegill sunfish 

Title: The Acute Toxicity of Cobratec TT 50S to the Bluegill Sunfish Lepomis macrochirus

Rafinesque

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 50% solution in water of the sodium salt of tolyltriazole (CAS No. 29385-43-1)

Remarks: Referred to as Cobratec TT 50S. Described as dark amber, water-soluble liquid.

METHOD

Method/guideline followed: Committee on Methods for Toxicity Tests with Aquatic 

Organisms (1975)

Test type: Acute LC50

GLP compliant?: Not specified

Year study was performed: 1979

Test species: Bluegill sunfish (Lepomis macrochirus)

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 

~ 0, 16.0, 28.0, 50.0, 90.0 and 160.0 mg sodium tolyltriazole/L

(0, 32.0, 56.0, 100.0, 180.0 and 320.0 mg Cobratec TT 50S / L)

Test method/conditions remarks: Dilution water for the test was obtained from a well on the 

Tarrytown site, treated with a Continental Model 3020 Reverse Osmosis Water System, and 

deionized; then, following procedures of Marking and Dawson (1973), it was reconstituted to a 

pH of 7.46, total hardness of 42 mg/L as CaCO3, total alkalinity of 30 mg/L as CaCO3 and 

specific conductance of 155 μmhos/cm. Hardness and alkalinity were determined according to 

standard analytical procedures (American Public Healthy Association, 1976), pH with an 

ORION pH Meter, conductivity with a YSI Conductivity Bridge and dissolved oxygen with a 

YSI Oxygen Meter.

Test practices followed these recommended by the Committee on Methods for Toxicity Tests 

with Aquatic Organisms (1975), except that replicate concentrations were not used. Samples of 

Cobratec TT 50S were weighed to a precision of 0.1 mg and added directly to the test containers. 
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Test vessels were 19.6 liter, chemically clean glass jars, each containing 15 liters of soft 

reconstituted dilution water and immersed in a constant temperature recirculating water bath. 

Testing was begun by adding measured quantities of test material to the water in five test vessels, 

and mixing thoroughly, to yield nominal concentrations of 32.0, 56.0, 100.0, 180.0 and 320.0 

mg/L. A sixth vessel, as control, contained 100% dilution water. Ten fish were introduced at 

random into each of the test and control vessels; these were approximately 4 months old with a 

mean (10 organisms) length of 37 mm and mean weight of 0.50 grams. Biological loading was 

0.33 g/L.

At the beginning of the test, and every 24 hours thereafter, dissolved oxygen and pH of the 

control and each test concentration, and temperature of the water bath, were determined. 

Mortalities among the test fish, and any observable abnormal behavioral responses, were noted 

and recorded every 24 hours. The no effect level was determined, by observation, at 96 hours.

The concentration of test material lethal to 50% of the test population, and 95% confidence 

limits, were determined for the 72- and 96-hour exposure periods by the Spearman-Karber 

Estimator. LC50s could not be calculated for the 24 and 48 hour exposure periods due to the lack 

of sufficient mortalities. LC50 calculations are based on nominal concentrations of the test 

material in soft reconstituted water.

RESULTS

Endpoint value(s): The 96-hour LC50 was ~ 95.6 mg sodium tolyltriazole/L (191.2 mg 

Cobratec TT 50S / L). The 96-hour NOEC was ~ 28.0 mg sodium tolyltriazole/L (56.0 mg

Cobratec TT 50S /L).

Results remarks: pH ranged from 7.18 to 9.4. Dissolved oxygen ranged from 5.2 to 9.3 mg/L.

CONCLUSIONS

None specified.

STUDY RELIABILITY

Klimisch code = [1]. Reliable without restriction.

REFERENCE

TSCATS submission (OTS0521126, Doc ID 86-890000618, received 06/13/89): Union Carbide 

Corp, Sherwin Williams Company. 1979. Initial submission: The Acute Toxicity of Cobratec 

TT 50S to the Bluegill Sunfish (Lepomis macrochirus) Rafinesque with Cover Letter Dated 

061289.
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Study 8: Acute toxicity study in rainbow trout 

Title: The Acute Toxicity of Cobratec TT50S to the Rainbow Trout Salmo gairdneri Richardson

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 50% solution in water of the sodium salt of tolyltriazole (CAS No. 29385-43-1)

Remarks: Referred to as Cobratec TT 50S. Described as dark amber, water-soluble liquid.

METHOD

Method/guideline followed: Committee on Methods for Toxicity Tests with Aquatic 

Organisms (1975)

Test type: Acute LC50

GLP compliant?: Not specified

Year study was performed: 1979

Test species: Rainbow trout (Salmo gairdneri) Richardson

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 

~ 0, 2.8, 5.0, 9.0, 16.0 and 28.0 mg sodium tolyltriazole / L

(0, 5.6, 10.0, 18.0, 32.0 and 56.0 mg Cobratec TT 50S/L)

Test method/conditions remarks: Dilution water for the test was obtained from a well on the 

Tarrytown site, treated with a Continental Model 3020 Reverse Osmosis Water System, and 

deionized; then, following procedures of Marking and Dawson (1973), it was reconstituted to a 

pH of 7.47, total hardness of 42 mg/L as CaCO3, total alkalinity of 28 mg/L as CaCO3 and 

specific conductance of 180 umhos/cm. Hardness and alkalinity were determined according to 

standard analytical procedures (American Public Healthy Association, 1976), pH with an 

ORION pH Meter, conductivity with a YSI Conductivity Bridge and dissolved oxygen with a 

YSI Oxygen Meter.

Test practices followed these recommended by the Committee on Methods for Toxicity Tests 

with Aquatic Organisms (1975), except that replicate concentrations were not used. A fresh 

working stock solution of the test material in deionized water was prepared by weight to a 

precision of 0.1 mg. Five concentrations were tested, with a control. Test vessels were 19.6 
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liter, chemically clean glass jars, each containing 15 liters of soft reconstituted dilution water and 

immersed in a constant temperature recirculating water bath.

Testing was begun by adding measured volumes of stock solution to the water in five test 

vessels, and mixing thoroughly, to yield nominal test material concentrations of 5.6, 10.0, 18.0, 

32.0 and 56.0 mg/L. A sixth vessel, as control, contained 100% dilution water. Ten fish were 

introduced at random into each of the test and control vessels; these were approximately 3 

months old with a mean (10 organisms) length of 54 mm and mean weight of 1.27 grams. 

Biological loading was 0.85 g/L.

At the beginning of the test, and every 24 hours thereafter, dissolved oxygen and pH of the 

control and each test concentration, and temperature of the water bath, were determined. 

Mortalities among the test fish, and any observable abnormal behavioral responses were noted 

and recorded every 24 hours. The no effect level was determined, by observation, at 96 hours. 

The concentration of test material lethal to 50% of the test population was determined for the 24, 

48, 72 and 96 hour exposure periods by the Spearman-Karber Estimator. Confidence limits for 

the 48 hour exposure period were also calculated. LC50 calculations are based on nominal 

concentrations of the test material in soft reconstituted water.

RESULTS

Endpoint value(s): The 96-hour LC50 was ~ 11.9 mg sodium tolyltriazole/L (23.7 mg Cobratec 

TT 50S /L). The 96-NOEC was ~ 5.0 mg sodium tolyltriazole/L (10.0 mg Cobratec TT 50S /L).

Results remarks: pH ranged from 7.03 to 8.42. Dissolved oxygen ranged from 6.2 to 9.4 

mg/L.

CONCLUSIONS

None specified.

STUDY RELIABILITY

Klimisch code = [1]. Reliable without restriction.

REFERENCE

TSCATS submission (OTS0521128, Doc ID 86-890000620, received 06/13/89): Union Carbide 

Corp, Sherwin Williams Company. 1979. Initial submission: The Acute Toxicity of Cobratec 

TT50S to the Rainbow Trout (Salmo gairdneri) Richardson with Cover Letter Dated 061289.
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B-2. ACUTE TOXICITY TO DAPHNIA

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna

Title: Comparative Measures of the Toxicity of Component Chemicals in Aircraft Deicing Fluid

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 5-Methyl-1H-Benzotriazole or 4-Methyl-1H-Benzotriazole (MeBT), CAS No. 29385-

43-1

Remarks: None

METHOD

Method/guideline followed: U.S. Environmental Protection Agency. 1985. Methods for 

measuring the acute toxicity of effluents and receiving waters to freshwater and marine 

microorganisms. EPA 600/4-85-013. Environmental Monitoring and Support Laboratory, 

Cincinnati, OH.

Test type: Acute EC50; Static

GLP compliant?: Not specified

Year study was performed: ~1999

Test species: Water fleas (Ceriodaphnia dubia) obtained from in-house culture.

Analytical monitoring: Not specified

Exposure duration: 48 hours

Concentrations tested: 152.1 and 183.8 mg/L

Test method/conditions remarks: All C. dubia used in the tests were <24 (±8) h old at test 

initiation. The toxicity of MeBT to C. dubia was assessed in moderately hard water using acute 

tests according to U.S. Environmental Protection Agency procedures. Mortality was the 

endpoint. The nominal (predilution) test concentrations of MeBT were 152.1 and 183.8 mg/L. 

A dilution water control was also included. Test chambers were 30-mL plastic cups. All tests 

were conducted at 20 oC with a light:dark cycle of 16:8 h. Testing was conducted under static 

conditions for a 48-hour duration. The LC50 values were calculated using either the graphical, 
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probit, Spearman-Karber, or trimmed Spearman-Karber methods according to U.S. 

Environmental Protection Agency-established criteria.

RESULTS

Endpoint value(s): The 48-hour EC50 value for C. dubia was 102-108 mg/L.

Results remarks: Measured high concentrations of MeBT were between 95.5 and 99.5% of 

nominal concentrations at a confidence interval of 95%. Lower test concentrations were 

calculated as a proportion of the 100% measured concentration. The LC50s were calculated 

based on measured 100% concentration.

CONCLUSIONS

The data provided by this study confirmed that MeBT contributes to the toxicological properties 

of aircraft deicing fluid formulations.

STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

Cornell, J.S. et al. 2000. Comparative measures of the toxicity of component chemicals in 

aircraft deicing fluid. Environmental Chemistry, 19(6): 1465-1472.
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Study 2: Acute toxicity study in Daphnia magna

Title: Static Acute Toxicity of RM 27513 to the Daphnid, Daphnia magna

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as RM 27513 (EnviroSystems Sample Number 2694E). Delivered on 

November 15, 1990. RM 27513 was supplied by 3M Company, 935 Bush Avenue, St. Paul, 

Minnesota. 

METHOD

Method/guideline followed: Static, Acute Toxicity Test With RM 27513 And The Daphnid, 

Daphnia magna. It is based on procedures of OECD (1984).

Test type: Acute static

GLP compliant?: Yes

Year study was performed: 1991

Test species: Daphnia magna

Analytical monitoring: No

Exposure duration: 48 hours

Concentrations tested: 0 (control), 15, 25, 40, 60, and 100 mg/L

Test method/conditions remarks:

Dilution water – Water used for acclimation of test organisms and for all toxicity testing was 

collected from wells at EnviroSystems in Hampton, New Hampshire. Water was stored in 500-

gallon polyethylene tanks where it was aerated. 

Test organism – Juvenile daphnids employed as test organisms were from a single source and 

were identified using an appropriate taxonomic key. Daphnids used in the test were produced 

from an in-house culture and were less than 24 hours old at the start of the test. Control daphnids 

were weighed at the conclusion of the toxicity test. Prior to testing, daphnids were maintained in 

100% dilution water under static conditions in 4 liter glass jars. During acclimation daphnids 

were not treated for disease and they were free of apparent sickness, injuries, and abnormalities 

at the beginning of the test. During the acclimation period 24 hours prior to the test initiation the 
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temperature was 19.0 – 19.8 oC. Daphnids were fed yeast, trout chow, and/or the freshwater alga 

Selenastrum capricornutum once or twice daily before the test.

Toxicity testing – A screening test with the test substance was conducted during November 27 to 

29, 1990. Nominal concentrations of test substance were: 0.1, 1.0, 10, 100, and 1000 mg/L. 

After 48 hours of exposure there was 0% survival at 1000 mg/L, 75% survival at 100 mg/L, and 

100% survival at all other tested concentrations.

The definitive toxicity test was performed during December 12 to 14, 1990, according to 

EnviroSystems Test Protocol 90169-3 (Static, Acute Toxicity Test With RM 27513 And The 

Daphnid, Daphnia magna), which was signed by the Study Director on November 16, 1990. It is 

based on procedures of the OECD (1984).

The test was conducted at a target temperature of 20 ± 1 oC with five concentrations of test 

substance and a dilution water control. A 1000 mg/L stock solution was prepared for combining 

1.0 g of test substance with dilution water and adjusting the final concentrations to 1000 ml 

without the use of a solvent. 

Twenty daphnids were randomly and equally distributed among four replicates of each 

treatment. The test was performed in 250 ml glass beakers that contained 200 ml of test solution 

(water depth was approximately 6 cm). Test vessels were randomly arranged in an incubator 

during the 48 hour test (a random numbers table was used to select the location of each vessel). 

A 16 hour light and 8 hour dark photoperiod was automatically maintained with cool-white 

fluorescent lights that provided a light intensity of 30 uEs-1

m-2. Aeration was not required to 

maintain dissolved oxygen concentrations above acceptable levels. Daphnids were not fed 

during the test.

The number of surviving organisms and the occurrence of sublethal effects and immobilization 

(inability to swim within 15 seconds after gentle agitation) or other sublethal effects (loss of 

equilibrium, erratic swimming, loss of reflex, excitability, discoloration, or change in behavior) 

were determined visually and recorded initially after 24 and 48 hours. Dead test organisms were 

removed when first observed. Dissolved oxygen, pH, conductivity and temperature were 

measured and recorded daily in each test chamber that contained live animals.

Results of the toxicity test were interpreted by standard statistical techniques (Stephan, 1983). 

Nominal concentrations of test substance the number of daphnids killed or immobilized were 

used to calculate the EC50 value.

RESULTS

Endpoint value(s): Exposure of daphnids to the test substance resulted in a 48-hour EC50 of 19 

mg/L Rm27513, with a 95% confidence interval of 12 – 25 mg/L. 24-hour EC50 = 34 mg/L

Results remarks: Test vessels containing RM 27513 appeared clear initially and remained free 

of insoluble material throughout the test. Ninety-five percent survival occurred in the control 

exposure. Control daphnids had an average wet weight (blotted dry) of 0.001 g at the end of the 

test. Loading rate during the toxicity test was approximately 0.02 g/L. Exposure of daphnids to 
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the reference toxicant sodium dodecyl sulfate resulted in a 48 hour median lethal concentration 

(LC50) of 13.8 mg/L.

pH ranged from 7.7 to 8.4 throughout testing. Conductivity ranged from 12000 to 13000 

μmho/cm. Temperature ranged from 19.0 to 20.7 oC. Dissolved oxygen ranged from 8.3 to 9.7 

mg/L.

CONCLUSIONS

None specified.

STUDY RELIABILITY

Klimisch code = [2] Reliable with restrictions.

REFERENCE

TSCATS submission (OTS0533466, Doc ID 86-920000103S, received 10/31/91): Resource 

Analysts, Inc., 3M Company. 1991. Initial submission: Environmental Effects of Tolyltriazole 

with Attachments and Cover Letter 102891 (SANITIZED). pp. 75-91.
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B-3. TOXICITY TO AQUATIC PLANTS

Summary of endpoint: Adequate

Study 1: Acute toxicity study in algae 

Title: Static Acute Toxicity of RM 27513 to the Alga, Selenastrum capricornutum

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as RM 27513 (EnviroSystems Sample Number 2694E). Delivered on 

November 15, 1990. RM 27513 (white granules) was supplied by 3M Company, 935 Bush 

Avenue, St. Paul, Minnesota. 

METHOD

Method/guideline followed: Not stated. Based on procedures of OECD (1984).

Test type: Acute static

GLP compliant?: Not stated

Year study was performed: 1991

Test species: Algae (Pseudokirchneriella subcapitata)

Analytical monitoring: No

Exposure duration: 96 hours

Concentrations tested: 0 (control), 62.5, 125, 250, 500, and 1000 mg/L

Test method/conditions remarks:

Dilution water – Water used for acclimation of test organisms and for all toxicity testing was 

sterile enriched media (U.S. EPA, 1978) with a pH of approximately 8.0. 

Test organism – Algae used for the test was from a culture originally procured from the Culture 

Collection of Algae at the University of Texas at Austin and delivered to Envirosystems on April 

12, 1990. The identity of the culture was verified using an appropriate taxonomic key. The 

culture was transferred to sterile enriched media identical to media used for this test and 

maintained at test conditions.
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Toxicity testing – The definitive toxicity test was performed during April 30 to May 4, 1991. It 

is based on procedures of the OECD (1984). The test was conducted at a target temperature of 

22 ± 2 oC with five concentrations of test substance and a dilution water control. A 1,000 mg/L 

stock solution was prepared in dilution water. The stock solution was added directly to dilution 

water contained in the test vessels without the use of a solvent. Algae were randomly and 

equally distributed among three replicates of each treatment at the rate of 10,000 cells/mL. The 

test was performed in 250 mL glass Erlenmeyer flasks that contained 100 mL of test solution. 

The flasks were capped with inverted glass beakers. Test vessels were randomly arranged on the 

rotary shaker in an incubatory during the 96 hour test (a random numbers table was used to select 

the location of each vessel). A 24-hour light and 0 hour dark photoperiod was maintained with 

cool-white fluorescent lights that provided a light intensity of 35 uEs-1m-2.

The number of algal cells/mL in each test vessel was determined visually every 24 hours by 

means of direct microscopic counts with a hemacytometer. The pH (Beckman model pHI 12 

meter) was determined in each test flask at the beginning and end of the test, and temperature 

(ASTM mercury thermometer) was measured and recorded daily in the incubator

Statistical methods – Results of the toxicity test were interpreted by standard statistical 

techniques (Stephan, 1983). The binomial or moving average method was used by the author to 

calculate EC50 values using nominal concentrations of test substance. The NOEC was calculated 

using Dunnett’s test, which includes a parametric one-way analysis of variance (ANOVA). The 

average specific growth rate was calculated as the natural log of the number of cells per ml at 96 

hours minus the natural log of the number of cells per ml at 0 hours divided by the exposure 

period. The percent change from control is calculated by subtracting the sample average specific 

growth rate from the control average specific growth rate, dividing that value by the average 

specific growth rate in the control and multiplying that value by 100. The percent change from 

control was used to compute EC50 and NOEC values.

RESULTS

Endpoint value(s): The 72-hour EC50 is <62.5 mg/L, and the 72-hour NOEC is 250 mg/L RM 

27513. The 96-hour EC50 is 87 mg/L and the 96-hour NOEC is 250 mg/L.

24-hour EC50 > 1000 mg/L

48-hour EC50 = 68 mg/L (95% confidence limits: 62-125 mg/L; binomial calculation) 

Results remarks: Temperature varied from 23.1 to 23.5 oC throughout testing. pH varied from 

6.7 to 9.4 throughout testing.

CONCLUSIONS

None specified

STUDY RELIABILITY

Klimisch code = [2] Valid with restrictions
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REFERENCE

TSCATS submission (OTS0533466, Doc ID 86-920000103S, received 10/31/91): Resource 

Analysts, Inc., 3M Company. 1991. Initial submission: Environmental Effects of Tolyltriazole 

with Attachments and Cover Letter 102891 (SANITIZED). pp. 30-44
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B-4. ACUTE TOXICITY

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats 

Title: Acute Oral LD50 Investigation in Rats of Cobratec TT 100 Sample No. 5459

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Cobratec TT 100 Sample No. 5459

METHOD

Method/guideline followed: Not Stated

Test type: Acute Oral

GLP compliant?: Not specified

Year study performed: ~1972

Test species: Rats (strain not stated)

Number of animals/sex/dose: 5/sex/dose

Route of administration: Oral

Vehicle: Not specified

Doses/concentrations tested: 502, 632, 795 and 1001 mg/kg

Statistical methods used: An LD50 value was calculated by the method of Litchfield and 

Wilcoxon.

Test method/conditions remarks: Cobratec TT 100 Sample No. 5459 was administered orally 

in graduated doses to 4 groups of 10 rats. Five males and five females in each group. They were 

observed for 14 days. 

RESULTS

Endpoint value(s): LD50 = 675 mg/kg (95% confidence limits were 535-851 mg/kg)
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Results remarks: Mortalities were as follows:

502 mg/kg – 3 deaths on day 0

632 mg/kg – 1 death on day 0; 2 deaths on day 1

795 mg/kg – 6 deaths on day 0; 1 death on day 1

1001 mg/kg – 10 deaths on day 0

CONCLUSIONS

None stated

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions

REFERENCE

TSCATS submission (Doc ID 86-890000263, received 06/05/89): Huntingdon Research Center, 

Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 19 Chemicals with 

Cover Letter Date 060289. pp. 32-34.
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Study 2: Acute oral toxicity study in rats 

Title: The Acute Oral Toxicity (LD50) to Rats of Olin 58734

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Olin 58734. Pinkish grey granular material

METHOD

Method/guideline followed: Method in Appraisal of the Safety of Chemicals in Food, Drugs, 

and Cosmetics, Association of Food and Drug Officials of the U.S. 1959

Test type: Acute oral

GLP compliant?: Not specified 

Year study performed: 

Test species: FDRL strain rats

Number of animals/sex/dose: 5/dose

Route of administration: Oral

Vehicle: Not specified 

Doses/concentrations tested: 500, 1000, 2000, 4000 and 8000 mg/kg-bw

Statistical methods used: Thompson moving average method

Test method/conditions remarks: Animals – Adult albino rats, FDRL strain, 5 rats per dose 

level

Observations – Body weight, behavior, appearance, mortality, gross changes at autopsy of rats 

that died and of representative survivors sacrificed at 14 days

Test substance was administered intragastrically as a 25% suspension in 0.5% (illegible).

Groups were comprised of three males and two females.
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RESULTS

Endpoint value(s): LD50 = 1830 mg/kg-bw

Results remarks: Deaths occurred in 3 hours to 5 minutes, following CNS depression, shallow 

breathing, lacrimation, and salivation. Liver congestion and pale stomach mucosae were seen at 

autopsy.

One mortality was observed at 0.5 g/kg; 0 mortalities at 1.0 g/kg; 3 mortalities at 2.0 g/kg; 5 

mortalities at 4.0 g/kg; 5 mortalities at 8.0 g/kg.

CONCLUSIONS

The acute oral LD50 for rats is 1.83 g per kg body weight; confidence limits = 1.20 to 2.81 g per 

kg body weight.

STUDY RELIABILITY

Klimisch code = [3] – Study is dated; lacks information on test substance

REFERENCE

TSCATS submission (OTS0520643, Doc ID 86-89000060A, received 06/13/89): Huntingdon 

Research Center, Sherwin Williams Co. 1968. Initial submission: Summary of Mucuous 

Membrane (Eye) Irritations in Rabbits Treated with Olin 58734 with Attachment and Cover 

Letter Dated 061289.
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Study 3: Acute inhalation toxicity study in rats 

Title: Evaluation of the Acute Inhalation Toxicity of Tolyltriazole

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Cobratec RTT 100, Sample No. 3000. The compound was a fine 

powder, off-white in color. The melting point was reported to be 76 oC, with rapid 

decomposition occurring at temperatures ≥ 300 oC.

METHOD

Method/guideline followed: Huntingdon Research Center’s Standard Design No. INH-SW1

Test type: Acute inhalation

GLP compliant?: Not specified

Year study performed: ~1976

Test species: Sprague-Dawley rats (250-400 grams)

Number of animals/sex/dose: 10/sex

Route of administration: Inhalation (aerosol)

Vehicle: Not specified

Doses/concentrations tested: nominal: 2 mg/L; actual: 1.73 ± 0.67 mg/L

Statistical methods used: Not stated

Test method/conditions remarks: One group of ten male and ten female Sprague-Dawley rats 

(Charles River) was exposed for one hour to a concentration of 1.73 ± 0.67 mg/L of an aerosol of 

tolyltriazole. Vapor phase compound was also monitored. Following the exposure, all animals 

were maintained for an additional fourteen days; with food and water ad libitum. Body weights 

were taken prior to exposure and at seven and fourteen days post-exposure. All animals were 

observed for signs of toxicity during the exposure and daily during the post-exposure observation 

period. All animals that died or were sacrificed after fourteen days were necropsied. At this 

time, all organs were observed for macroscopic abnormalities. The following tissues were stored 

in 10% neutral buffered formalin in the event that histopathology might be required: lungs and 

trachea, heart, liver, kidneys, spleen, stomach, intestines, gonads, whole head, abnormal tissue.
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Animals were allowed 7 days to accommodate to the laboratory conditions before being assigned 

to the study.

Aerosol generation system: Approximately one hundred grams of tolyltriazole is placed in the 

round bottom three necked flask and melted. The temperature of the flask and delivery tube is 

maintained at 200 oC by the heating mantle, air heater and heating tape. A controlled air flow of 

ten L/min is provided to the vapor generating flask. The tolyltriazole vapor-laden air is taken 

from the top of the generating flask, diluted and cooled by admixture of a controlled flow of 

clean dry air. This system provided a very fine white aerosol. Chamber concentration was 

controlled by varying the air flow rate through the generator.

Exposure concentrations were determined by sampling the chamber atmosphere. Actual 

exposure concentrations were determined by drawing (at 0.82 L/min) a known volume of 

chamber air through a pre-weighted glass fiber filter in a closed face filter holder. The sample 

time was three minutes. The filter was then re-weighed and the chamber concentration 

calculated.

Vapor phase compound was determined by sampling the chamber atmosphere. The sampling 

rate was 0.82 L/min for six minutes. The sample was drawn through two 25 mL fritted gas 

bubblers in series, each containing 10 mL of 0.5N sodium hydroxide. After sample collection 

the contents of each gas bubbler was transferred to a 25 mL volumetric flask, brought to volume 

with additional 0.5N sodium hydroxide and mixed well. The absorbance was determined at 274 

nm using a UV spectrophotometer and matched 1 cm. silica cells. The tolyltriazole content was 

obtained by reference to a standard curve.

Particle size analysis was also conducted during the exposure. An Anderson cascade impactor 

was used for this determination. Pre-weighted glass fiber filters, specially cut to fit the impactor, 

were used for collecting the aerosol on each stage. The filter discs were re-weighed immediately 

after sampling. A particle size distribution graph was then constructed from the weight of 

material collected on each stage. The aerosol was found to have an Equivalent Aerodynamic 

Diameter (EAD) of 0.35 μm with a geometric standard deviation of 2.62. The sample flow rate 

was 1 CFM for ten minutes.

RESULTS

Endpoint value(s): LC50 > 1.73 mg/L

Results remarks: During the exposure, the only pharmacologic or toxicologic sign observed 

was a decrease in general activity. By 24 hours post-exposure, all animals were apparently 

normal and remained so during the entire 14-day observation period. All surviving animals 

appeared to gain weight normally throughout the 14 day observation period.

Since there were no deaths during the exposure or 14-day post-exposure observation period, all 

animals were necropsied following the 14-day observation period. All organs were observed for 

gross abnormalities. Respiratory tract involvement, characterized by slight to moderate 
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incidence of hemorrhage or hepatization, was found in all animals. Some incidence of pale liver 

and kidneys was also noted in males and females.

CONCLUSIONS

None specified

STUDY RELIABILITY

Klimisch code = [1]. Valid without restriction

REFERENCE

TSCATS submission (OTS0521119, Doc ID 86-890000610, received 06/13/89): Huntingdon 

Research Center, Sherwin Williams Co. 1976. Initial submission: Evaluation of the Acute 

Inhalation Toxicity of Tolyltriazole (Final Report) with Attachments and Cover Letter Dated 

061289.
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Study 4: Acute dermal toxicity study in rabbits 

Title: Report on Acute Dermal Toxicity Investigation in Rabbits of Cobratec TT-100 

(Tolyltriazole) Sample No. 6022

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Substance was labeled Cobratec TT-100 (Tolyltriazole) Sample No. 6022. The 

material was light tan granular in nature.

METHOD

Method/guideline followed: Not stated

Test type: Acute dermal toxicity

GLP compliant?: Not specified

Year study performed: ~1972

Test species: Rabbits (strain not specified)

Number of animals/sex/dose: 10 females

Route of administration: Dermal

Vehicle: Not specified

Doses/concentrations tested: 2000 mg/kg

Statistical methods used: Not stated

Test method/conditions remarks: The material was applied to the clipped skin of 5 female 

rabbits and to the clipped and abraded skin of 5 female rabbits. The material was administered 

and allowed to remain in contact with the skin for 24 hours. The trunk of each animal was 

wrapped with a dam. After 24 hours the dams were removed. The residue was washed off with 

water. All rabbits were observed for mortality and toxic effects on the day of application and 

daily thereafter until death occurred or the 14 day observation period ended.

RESULTS

Endpoint value(s): LD50 > 2000 mg/kg
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Results remarks: No mortalities were observed. Body weights remained stable throughout the 

study (varying from 2.8 to 3.1 kg). Very slight erythema (grade 1) was observed in three rabbits, 

each from the abraded group; these effects subsided after the third day of observation. No 

additional signs of skin irritation were observed.

CONCLUSIONS

None stated

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions

REFERENCE

TSCATS submission (OTS0516797, Doc ID 86-890000263, received 06/05/89): Huntingdon 

Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 

19 Chemicals with Cover Letter Date 060289. pp. 10-15. 
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Study 5: Acute dermal toxicity study in rabbits 

Title: The Acute Dermal Toxicity to Rabbits of Olin 58734

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Olin 58734. Pinkish grey granular material

METHOD

Method/guideline followed: Method in Appraisal of the Safety of Chemicals in Foods, Drugs 

and (illegible) Association of Food and Drug Officials of the U.S. 1959

Test type: Acute dermal

GLP compliant?: Not specified

Year study performed: 

Test species: Albino rabbits

Number of animals/sex/dose: 2/sex/dose

Route of administration: Dermal

Vehicle: Not specified 

Doses/concentrations tested: 500, 1000, 2000 and 4000 mg/kg-bw

Statistical methods used: Not stated

Test method/conditions remarks: Animals – Four albino rabbits per four levels in weight range 

of 2.2 to 3.6 kg depilated over the trunk.

Maintenance conditions – Trunk encased in sleeve with animal restrained in stock during 

application. Thereafter individual housing with food and water ad libitum.

Dosage – 24 hour continuous contact with the skin. Test substance was administered as a 40% 

aqueous paste

Observations – Body weight, appearance, behavior, skin irritation and mortality over a 14-day 

period
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RESULTS

Endpoint value(s): LD50 > 4000 mg/kg-bw

Results remarks: No deaths occurred at any level in 14 days. The primary irritation scores were 

1.4, 1.5, 1.1 and 1.4, for the 0.5, 1, 2 and 4 g/kg levels, respectively. No other noteworthy 

findings were observed.

CONCLUSIONS

The acute dermal toxicity of the tested substance is greater than 4 g/kg-bw and this product 

evokes no irritation.

STUDY RELIABILITY

Klimisch code = [3] – Lack of study details; study is outdated

REFERENCE

TSCATS submission (OTS0520643, Doc ID 86-89000060A, received 06/13/89): Huntingdon 

Research Center, Sherwin Williams Co. 1968. Initial submission: Summary of Mucuous 

Membrane (Eye) Irritations in Rabbits Treated with Olin 58734 with Attachment and Cover 

Letter Dated 061289.
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B-5. SKIN IRRITATION

Study 1: Skin irritation study in rabbits 

Title: Primary Skin Irritation Study on the Rabbit of Cobratec TT-100 (Tolyltriazole) Sample 

No. 6022

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Cobratec TT-100 Sample No. 6022

METHOD

Method/guideline followed: Not stated

Test type: Skin Irritation

GLP compliant?: Not stated

Year study performed: ~1972

Test species: New Zealand albino rabbits

Number of animals/sex/dose: 12 females

Route of administration: Dermal

Vehicle: Not specified

Doses/concentrations tested: 0.5 grams

Statistical methods used: Not stated

Test method/conditions remarks: Twelve adult female New Zealand albino rabbits were used. 

Prior to application the backs were clipped free of hair. The skin was left intact on six of the 

rabbits and was abraded on the other six. One half gram of the sample was applied to an area of 

skin approximately one inch square, two single layers thick, were applied over the samples and 

covered with a non-reactive tape. The rabbits were then wrapped with a binder.

After the 24-hour exposure, the binders and the tape were removed and the skin reactions 

evaluated. An evaluation was also made at 72 hours after application.

The reactions were scored according to the method described in the Federal Hazardous 

Substances Act.
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RESULTS

Endpoint value(s): Not irritating. Scores of 0 for erythema and edema at 24 and 72 hours for all 

rabbits.

Results remarks: The primary irritation index was 0.0. There was no skin irritation.

CONCLUSIONS

Not irritating to rabbit skin.

STUDY RELIABILITY

Klimisch code = [2] - Valid with restrictions

REFERENCE

TSCATS submission (OTS0516797, Doc ID 86-890000263, received 06/05/89): Huntingdon 

Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 

19 Chemicals with Cover Letter Date 060289. pp. 51-57.
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B-6. EYE IRRITATION

Study 1: Eye irritation study in rabbits 

Title: Eye Irritation Study in the Rabbit of Tolyltriazole (Cobratec TT-100) Sample #1089

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Cobratec TT-100 Sample No. 1089

METHOD

Method/guideline followed: Not stated

Test type: Eye Irritation

GLP compliant?: Not stated

Year study performed: ~1972

Test species: New Zealand rabbit

Number of animals/sex/dose: 6 females

Route of administration: Ocular

Vehicle: Not specified

Doses/concentrations tested: 100 mg

Statistical methods used: Not stated

Test method/conditions remarks: Six female albino New Zealand rabbits were used. Both 

eyes of each animal were examined with the aid of an ophthalmoscope before testing. 100 mg of 

test material was placed in one eye of each animal by gently pulling the lower lid away from the 

eyeball to form a cup into which the test substance was dropped. The lids were gently held 

together for one second and the animal released. The other eye, remaining untreated, served as a 

control.

The eyes were examined and the grade of ocular reaction recorded at 24, 48, 72 hours and 14 

days. Reading of reactions was facilitated by the use of an ophthalmoscope. After the recording 

of observations at 24 hours, the eyes were stained with fluorescein and examined with an ultraviolet lamp to detect corneal damage if present.
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The reactions were scored from: “Illustrated Guide for Grading Eye Irritation by Hazardous 

Substances,” Section 191.12 of the Federal Hazardous Substances Labeling Act Regulations. 

RESULTS

Endpoint value(s): The test substance was found to be irritating to rabbit eyes. Conjunctival 

irritation scores ranged from 0 to 4 throughout the observation period. Corneal irritation scores 

ranged from 0 to 3. Iridial irritation scores ranged from 0 to 1.

Results remarks: One rabbit died prior to the 14 day reading. All rabbits exhibited treatmentrelated effects on the conjunctiva and cornea. 4/6 rabbits exhibited treatment-related effects on 

the iris. 

No. Observed / No. Exposed

Cornea observations –

2/6 – a diffuse area of corneal opacity

3/6 – discernible translucent areas of opacity, details of iris slightly obscured

1/6 – no details of iris visible

6/6 – fluorescein examination positive

Iris observations – 4/6 – moderate circumcorneal injection

Conjunctival redness –

4/6 – diffuse crimson red

2/6 – diffuse beefy red

Chemosis –

1/6 – swelling with partial eversion of lids

2/6 – swelling with lids about half closed

3/6 – swelling with lids half closed or greater

CONCLUSIONS

Irritating to rabbit eyes

STUDY RELIABILITY

Klimisch code = [2] - Valid with restrictions

REFERENCE

TSCATS submission (Doc ID 86-890000263, received 06/05/89): Huntingdon Research Center, 

Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 19 Chemicals with 

Cover Letter Date 060289. P 60-65.
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Study 2: Eye irritation in rabbits study

Title: Summary of Mucous Membrane (Eye) Irritation in Rabbits Treated with Olin 58734

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as Olin 58734. Pinkish grey granular material

METHOD

Method/guideline followed: Method in Appraisal of the Safety of Chemicals in Food, Drugs, 

and Cosmetics, Association of Food and Drug Officials of the U.S. 1959, p. 46

Test type: Eye Irritation

GLP compliant?: Not stated

Year study performed:

Test species: Albino rabbits

Number of animals/sex/dose: 3/sex/dose

Route of administration: Ocular

Vehicle: Not specified 

Doses/concentrations tested: 10 mg

Statistical methods used: Not stated

Test method/conditions remarks: Dosage – 10 mg of powdered sample in right eye, without 

washout.

Maintenance conditions – Animals maintained in stocks for 6 hours after dosage. Thereafter, 

individual housing with food and water ad libitum.

Observations – Evaluation of degree of eye irritation at 24, 48, and 72 hours, as described in 

Regulations

RESULTS

Endpoint value(s): Slightly to transiently irritating
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Results remarks: Cornea – Moderate opacity in small area in 3 on day 1, in 2 on day 2, in 1 on 

day 3.

Iris – Irritation in 1 on days 1, 2, and 3.

Conjunctiva – Lid redness slight in 6 on day 1, very slight in 2 on day 3. Chemosis slight in 4 on 

day 1. Discharge slight in 6 on day 1, very slight in 1 on day (illegible).

CONCLUSIONS

The test substance is slightly and transiently irritating to the eyes of rabbits.

STUDY RELIABILITY

Klimisch code = [2] - Valid with restrictions

REFERENCE

TSCATS submission (OTS0520643, Doc ID 86-89000060A, received 06/13/89): Huntingdon 

Research Center, Sherwin Williams Co. 1968. Initial submission: Summary of Mucous 

Membrane (Eye) Irritations in Rabbits Treated with Olin 58734 with Attachment and Cover 

Letter Dated 061289.
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B-7. SKIN SENSITIZATION

Study 1: Skin senitization study in guinea pigs 

Title: Report on Skin Sensitizing (Contact Allergenic) Effect in Guinea Pigs of TK 10 637/1

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as TK 10 637/1, Lot 1179/1186, 97.8% pure

METHOD

Method/guideline followed: The optimization test

Maurer Th., Thomann P., Weirich E.G., Hess R.

The optimization test in the guinea pig.

Agents & Actions 5(2), 174-179, 1975.

Predictive evaluation in animals of the contact allergenic potential of medically important 

substances.

Contact Dermatitis 4, 321-333, 1978.

Contact Dermatitis 5, 1-10, 1979.

The optimization test in the guinea pig in relation to other predictive sensitization methods. 

Toxicology 15, 163-171, 1980.

Test type: Skin sensitization

GLP compliant?: Not stated

Year study performed: 1982

Test species: Pirbright white guinea pigs

Number of animals/sex/dose: 10/sex

Route of administration: intradermal/epidermal

Vehicle: Saline

Doses/concentrations tested: 0.1% for intradermal application

10% for epidermal application

Statistical methods used: Not stated

Test method/conditions remarks: The optimization test was used, an intracutaneous 

sensitization procedure exceeding the sensitivity of the method recommended in the “Appraisal 
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of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959), the US Association of Food 

and Drug Officials (AFDO).

The test was performed on 10 male and 10 female guinea pigs of the Pirbright white strain, bred 

on the premises and weighing between 221 to 431 grams (~10 weeks old). 

The animals were housed individually in Macrolon cages type 3, assigned to the different groups 

by means of random numbers generated by the random number generator incorporated in the 

Hewlett-Packard desk computer, identified with individual ear tags, kept at a constant room 

temperature of 20 ±1 C, at a relative humidity of 50 ± 10% and on a 14 hours light cycle. The 

animals received ad libitum standard guinea pig pellets and water, supplemented with fresh 

carrots. The animals were acclimatized for 12 days. 

After the 24-hour exposure, the binders and the tape were removed and the skin reactions 

evaluated. An evaluation was also made at 72 hours after application. The reactions were scored 

according to the method described in the Federal Hazardous Substances Act.

During the induction period the animals received one injection every second day (except 

weekends) to a total of 10 intracutaneous injections of a freshly prepared 0.1% solution of TK 10 

637/1 in physiological saline. One control group was treated with the vehicle alone (“negative 

control”).

On the first day, injections of 0.1 mL were administered into the shaved skin of the right flank 

and the back, while on the following days a single intracutaneous injection was given into the 

back. 

During the second and third week of the induction period the test material was incorporated in a 

mixture of the normal vehicle with complete Bacto Adjuvant.

(vehicle:adjuvant = 1:1).

Fourteen days after the last sensitizing injection, a challenge injection of 0.1 mL of a freshly 

prepared 0.1% solution of TK 10 637/1 in physiological saline was administered into the skin of 

the left flank. 

Twenty-four hours after each injection during the first week of the induction period and 24 hours 

after the challenge injection the reactions were recorded.

The two largest perpendicular diameters (in mm) and the increase in the skin-fold thickness (in 

mm) were measured and by multiplication of these values a “reaction volume” was obtained (in 

μl) for each reading from each animal. The mean volume plus one standard deviation of the 

induction reactions observed in the individual animal in the first week was taken as representing 

the skin irritation “threshold” for each animal. Any challenge reaction greater than this threshold 

value in the induction period was graded as an allergic reaction and the animal termed “positive”. 

The number of “positive” animals in the test group was compared with the number of animals in 

the control group (treated with the vehicle alone) that showed a non-specific reaction of at least 

the same magnitude (“negative control”).
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Ten days after the intracutaneous challenge injection a subirritant dose of the test compound 

(10% TK 10 637/1 in vaseline) was applied epicutaneously under occlusive dressings which 

were left in place for 24 hours. The reactions were evaluated 24 hours after removing of the 

bandages according to Draize scoring scale.

RESULTS

Endpoint value(s): No animals were sensitized following exposure to TK 10 637/1. All 

erythema scores were zero on Draize scale.

Results remarks: Under the experimental conditions employed, no differences between the test 

group and the vehicle-treated controls were seen, after either intradermal or epidermal challenge 

application of TK 10 637/1. TK 10 637/1 was found to be devoid of skin-sensitizing (contact 

allergenic) potency in albino guinea-pigs.

CONCLUSIONS

Not a sensitizer

STUDY RELIABILITY

Klimisch code = [1]

REFERENCE

TSCATS submission (OTS0519042, Doc ID 86-890000273, received 06/07/89): Ciba Geigy 

Corp. 1982. Initial submission: Report on Skin Sensitizing Effect in Guinea Pigs of TK 10 

637/1 with Cover Letter Dated 060589.
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B-8. GENE MUTATION

Summary of endpoint: Adequate

Study 1: In vitro mutation in Salmonella study

Title: A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella 

typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: None

METHOD

Method/guideline followed: As described in Ames, B. N., McCann, J., and Yamasaki, E. (1975) 

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome

mutagenicity test. Mutat. Res. 31, 347-364.

Test type: Ames assay

GLP compliant?: Not specified

Year study performed: Not stated

Test species: Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, 

and TA1538 were obtained from Dr. Bruce Ames, University of California (Berkeley, CA). 

Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation 

when they reached a density of (1-2) _ 109 cells/mL. On the day of use, all tester strain cultures 

were checked for genetic integrity as recommended by Ames et al.

Metabolic activation: With and without

Concentrations tested: 100 – 10,000 μg/plate

Statistical methods used: Not stated

Test method/conditions remarks: The experimental materials, methods, and procedures as 

explained below are based on those described by Ames et al.. For initial mutagenicity testing, the 

plate incorporation assay was used; some compounds were also tested using the flavin 

mononucleotide (FMN)-dependent reducing conditions described by Prival and Mitchell to break 

the diazo bonds of the condensed dyes, forming the individual constituents.
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S9 Preparation. Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian 

golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The

postmitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al. 

(26). The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-

phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 

homogenate at a concentration of 0.1 mL/mL of mix. For each plate receiving microsomal 

enzymes, 0.5 mL of S9 mix was added.

Plate Incorporation Methodology. For testing in the absence of S9 mix, 100 μL of the tester 

strain and 50 μL of the solvent or test chemical were added to 2.5 mL of molten selective top 

agar at 45 ±2 °C. When S9 was used, 0.5 mL of S9 mix, 50 μL of tester strain, and 50 μL of 

solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ±2 °C. After it 

was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After 

the overlay had solidified, the plates were incubated for 48 h at 37 ±2 °C. Five doses of test 

chemical, together with the appropriate concurrent solvent and positive controls, were tested in 

triplicate on each tester strain without metabolic activation and also with activation by induced

rat and hamster liver S9 preparations.

The doses that were tested in the mutagenicity assay were selected based on the levels of 

cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose 

levels of the chemical, one plate per dose, were tested in both the presence and the absence of 

induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test 

chemical per plate was used. The criteria used to evaluate a test were as follows: for a

test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and 

TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in

the mean revertants per plate had to be accompanied by a dose response to increasing 

concentrations of the test chemical. If the study showed a dose response with a less than 3-fold 

increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.

Preincubation Methodology under Reductive Conditions. For tests using the FMN-modified 

assay, strains TA98 and TA100 were used. The bacteria, uninduced hamster liver S9 (30% v/v),

cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and 

test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen 

was blown over the preincubation tube to keep the atmosphere reduced. At the end of the 

incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was 

poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by 

Ames et al. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN

experiments was Congo red. All plates were counted with an Artek automated colony counter 

(Artek 880, DynaTech, Chantilly, VA) or Minicount colony counter (Imaging Products 

International, Inc., Chantilly, VA), which was calibrated prior to use.

RESULTS

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not stated
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Results remarks: None specified

CONCLUSIONS

None specified

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions

REFERENCE

Seifried et al. 2006. Compilation of two decades of mutagenicity test results with the Ames 

Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays. Chem. Res. 

Toxicol. 19: 627-644.
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Study 2: In vitro mutation in Salmonella study

Title: Results from the testing of 300 chemicals

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: 99+ % pure

METHOD

Method/guideline followed: NTP Preincubation Protocol

Test type: Salmonella

GLP compliant?: Not specified 

Year study performed:1983

Test species: Bacterial Strains – Salmonella typhimurium strains TA97, TA98, TA100, TA1535 

and TA1537 were obtained from Dr. Bruce Ames (University of California, Berkeley) and were 

stored as recommended [Maron and Ames, 1983]. Cultures were grown overnight with shaking 

at 37 oC in Oxoid No. 2 broth, and their phenotypes were analyzed prior to their use for 

mutagenicity assays.

Metabolic activation: With and without

Concentrations tested: 0, 33, 100, 333, 1000, 1666, 3333, 6666 μg/plate

Statistical methods used: Not specified

Test method/conditions remarks: The vehicle control was dimethylsulfoxide.

Preparation of Liver S-9 Fractions – The S-9 (9,000 g supernatant) fractions of Aroclor 1254-

induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described 

previously [Haworth et al, 1983]. The S-9 mixes were prepared immediately prior to use and 

contained either 10% or 30% S-9; occasionally, other levels were used. All chemicals were 

tested in the absence of metabolic activation and with rat and hamster S-9 fractions.

Test Protocol – The preincubation assay was performed as described previously [Haworth et al., 

1983], with some differences, as described below. The test chemical (0.05 mL), Salmonella 

culture (0.10 mL), and S-9 mix or buffer (0.50 mL) were incubated at 37 oC, without shaking, for 

20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top 

agar was added and the contents of the tubes were mixed and poured onto the surface of petri 
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dishes containing Vogel-Bonner medium [Vogel and Bonner, 1956]. The histidine-independent 

(his+) colonies arising on these plates were counted following two days incubation at 37 oC. 

Plates were machine counted unless precipitate was present which interfered with the count, or 

the color of the test chemical on the plate reduced the contrast between the colonies and the 

background agar. At the discretion of the investigators, plates with low numbers of colonies 

were counted by hand.

Variations in the protocol among the tested chemicals reflect the evolution of the protocol 

originally described by Haworth et al. [1983]. Four protocol variations are evident. 1) Testing 

in strains TA97, TA98, TA100 and TA1535, with some additional testing in strain TA1537; 10% 

S-9 was used. 2) The first test of a chemical was without activation and with 10% S-9 in the 

mix. If a positive result was obtained the test was repeated. If the tests were negative they were 

repeated without S-9 and with 30% S-9. 3) The order of use of 10% and 30% S-9 was reversed. 

4) Initial testing was in strains TA98 and TA100 without activation and with 30% rat and 

hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the 

other strains were not used. If the tests were negative, the other strains were used with 30% and 

10% S-9. A chemical was not designated nonmutagenic unless it had been tested in strains 

TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10% and 30% 

rat and hamster S-9. Occasionally, 5% S-9 was also used in all protocol variations.

All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for 

the mutagenicity assay. The toxicity assay was performed using TA100 or the system developed 

by Waleh et al. [1982]. Toxic concentrations were those that produced a decrease in the number 

of his+ colonies, or a clearing in the density of the background lawn, or both.

Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or 

to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent 

trials occasionally used narrower dose increments and may not have included doses in the toxic 

range. Experiments were repeated at least one week following the initial trial. A maximum of 

0.05 mL solvent was added to each plate.

Concurrent solvent and positive controls were run with each trial. The positive controls in the 

absence of metabolic activation were sodium azide (TA1535 and TA100), 9-aminoacridine 

(TA97 and TA1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for 

metabolic activation with all strains was 2-aminoanthracene. Occasionally, a laboratory used a 

single solvent and/or positive control for more than one chemical tested on the same day. Each 

laboratory made its own determination regarding positive control dose levels.

A chemical was judged mutagenic or weakly mutagenic if it produced a reproducible doserelated response over the solvent control in replicate trials. A chemical was judged questionable 

if the results of the individual trials were not reproducible, if increases in his+ revertants did not 

meet the criteria for a ‘weakly mutagenic’ response, or if only single doses produced increases in 

his+ revertants in repeat trials. Chemicals were judged nonmutagenic if they did not meet the 

criteria for a mutagenic or questionable response. 
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RESULTS

Genotoxicity result: Weak positive

Cytotoxic concentration (if applicable): Toxicity was observed at 6666 μg/plate in TA97 with 

activation and in TA1535 with activation.

Results remarks: None indicated.

CONCLUSIONS

Weakly Positive

STUDY RELIABILITY

Klimisch code = [1]. Reliable without restriction.

REFERENCE

NTP study 651117. Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. 

Salmonella mutagenicity tests. IV. Results from the testing of 300 chemicals Environ. Molec. 

Mutagen. Vol. 11 (Suppl 12) (1988) 1-158
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Study 3: In vitro mutation in Salmonella study

Title: Determination of Mutagenic/Carcinogenic and Cytotoxic Potential of Four Chemical 

Compounds

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Code Identification – TT-100; Sample Number – X18BT7565 Cobratec 78669A; 

Description – Tan round crystals

METHOD

Method/guideline followed: Not stated

Test type: Ames assay

GLP compliant?: Not specified 

Year study performed: ~1978

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, 

obtained directly from Ames.

Metabolic activation: With and without

Concentrations tested: 0, 80 μg, 400 μg, 2 mg, 10 mg

Statistical methods used: Not stated

Test method/conditions remarks: Dimethylsulfoxide (DMSO) was used to prepare stock 

dilutions of test materials and positive control chemicals. Approximately 108 cells from an 

overnight culture of each indicator strain (TA1535, TA1537, TA1538, TA98, TA100) were 

added to separate test tubes containing 2.0 mL of molten agar supplemented with a trace of 

histidine and biotin. For nonactivation tests, three dose levels of the extract were added to the 

content of the appropriate tubes and poured over the surface of selective agar plates. In 

activation tests, the extract concentrations were likewise added to the appropriate tubes with 

cells. Then, just prior to pouring, an aliquot of the S-9 mix containing the rat liver microsomes 

was added to each of the top agar tubes which were then mixed and the contents poured over the 

surface of a minimal agar plate and allowed to solidify. The plates were incubated for 48 hours 

at 37 oC, and scored for the number of colonies growing on each plate. 

Positive control/strain/concentration (μg/plate)
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Nonactivation

Sodium azide / TA1535 / 10

Quinacrine / TA1537 / 200

2-nitrofluorene / TA1538, TA98, TA100 / 10

Activation

2 amino anthracene / TA1535, TA1537 / 10

2 amino fluorine / TA1538, TA98, TA100 / 10

RESULTS

Genotoxicity result: Equivocal

Cytotoxic concentration (if applicable): 10 mg

Results remarks: The initial data obtained by the mutagenic bioassay of compound TT-100 

suggested a possible response with tester strains TA1538 and TA98. Confirmatory assays were 

carried out two additional times. The results do not clearly indicate that TT-100 is a frameshift 

mutagen. The revertant response elicited at the highest concentration tested indicated a slight 

elevation of the revertant rate but did not produce a response of at least an excess of two times 

background. Higher concentrations of the test compound indicated a toxic response. It appears 

that the TT-100 may possess some very weak mutagenic activity in this system when 

enzymatically activated; however, confirmation of this activity is negated by the toxicity of the 

compound in this system.

For the initial assay, in the absence of metabolic activation, toxicity was observed at 10 mg in all 

strains tested. In the presence of metabolic activation, toxicity was observed at 10 mg in 

TA1535 and TA1537.

For the confirmation assay, toxicity was observed with metabolic activation at 10 mg for 

TA1538 and TA98.

Positive and negative controls responded appropriately.

CONCLUSIONS

None stated

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions

REFERENCE

TSCATS submission (OTS0516797, Doc ID 86-890000263, received 06/05/89): Huntingdon 

Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 

19 Chemicals with Cover Letter Date 060289. pp. 72-91.
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Study 4: In vitro mutation in Salmonella study

Title: Salmonella/Mammalian-Microsome Mutagenicity Test with TK 10 637/1

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Referred to as TK 10 637/1

METHOD

Method/guideline followed: Ames, B.N., F.D. Lee, and W.E. Durston (1973), An Improved 

Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens.

Proc. Natl. Acad. Sci. USA 70, 782-786.

Test type: Ames assay

GLP compliant?: Not specified 

Year study performed: 1982

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538

Metabolic activation: With and without

Concentrations tested: 25, 75, 675, 2025 μg/0.1 mL without activation and 500, 1000, 2000, 

4000, 8000 μg/0.1 mL with activation.

Statistical methods used: Not stated

Test method/conditions remarks: The substance was dissolved in acetone. Acetone alone 

was used for the negative controls. Each Petri dish contained: 1) approx. 20 mL of minimum 

agar, 2) 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial 

culture in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6% agar solution 

with 0.6% NaCl and 10 mL of a solution of l-histidine, 0.5 mM and +biotin 0.5 mM. In the 

experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture 

was added also. 1 mL activation mixture contained: 0.3 mL S9 fraction of liver from rats 

induced with Aroclor 1254 and 0.7 mL of a solution of co-factors. 

Positive control experiments were carried out simultaneously with the following substances: 1) 

for strain TA98: daunorubicin-HCl, 5 and 10 μg/0.1 mL phosphate buffer; 2) for strain TA100: 

4-nitroquinoline-N-oxide, 0.125 and 0.25 ug/0.1 mL phosphate buffer; 3) for strain TA1535: Nmethyl-N’-nitro-N-nitrosoguanidine, 3 and 5 μg/0.1 mL phosphate buffer; 4) for strain TA1537: 

9(5)aminoacridine hydrochloride monohydrate, 50 and 100 μg/0.1 mL DMSO; 5) for strain 
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TA1538: 2-nitrofluorene, 5 and 10 μg/0.1 mL DMSO. The activation mixture was tested with 

strain TA1535 and cyclophosphamide, 250 μg/0.1 mL phosphate buffer and with strain TA98 

and 2-aminoanthracene, 5 μg/0.1 mL DMSO.

In the experiments without and with the addition of microsomal activation mixture three Petri 

dishes were prepared per strain and per group (i.e. per concentration or per control group).

The plates were incubated for about 48 hours and 37 oC in darkness.

When the colonies bad been counted, the arithmetic mean was calculated. The test substance is 

generally considered to be non-mutagenic if the colony count in relation to the negative control 

is not doubled at any concentration.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not specified

Results remarks: In the experiments performed without microsomal activation, none of the 

tested concentrations of TK 10 637/1 led to an increase in the incidence of histidine-prototrophic 

mutants comparison with the negative controls.

In the experiments with microsomal activation treatment with TK 10 637/1 led to an increase in 

the number of back-mutant colonies of strains TA98 and TA1538 at the concentrations of 2000 

ug/0.1 mL and above.

CONCLUSIONS

None stated

STUDY RELIABILITY

Klimsch code = [1] Reliable without restriction.

REFERENCE

TSCATS submission (OTS0519041, Doc ID 86-890000272, received 06/07/89): CIBA GEIGY 

Corp. 1982. Initial submission: Salmonella/Mammalian – Microsome Mutagenicity Test with 

TK 10 637/1 with Cover Letter Dated 060589.
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Study 5: In vitro mouse lymphoma cell mutation assay

Title: A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella 

typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assays

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: None

METHOD

Method/guideline followed: Not specified

Test type: L5178Y Mouse Lymphoma Cell Mutation Assay

GLP compliant?: Not specified 

Year study performed: Not specified

Test species/strain: L5178Y TK+/- 3.7.C mouse lymphoma cells

Metabolic activation: With and without

Concentrations tested: 12.5 – 750 μg/mL

Statistical methods used: Not specified

Test method/conditions remarks: L5178Y TK+/- 3.7.C mouse lymphoma cells were 

Originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research

Triangle Park, NC). The cells were grown in Fischer’s medium for leukemic cells of mice 

(Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10%

horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., 

Wyandotte, MI). Cells were screened for the presence of mycoplasma after cryopreservation. 

New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.

The toxicity of each chemical was determined both with and without liver S9 prepared from 

Aroclor 1254-induced male Sprague-Dawley rats. S9 mix was prepared according to the 

procedure of Clive et al. Cells at a concentration of 6 x 105

/mL (6 x 106 cells total) were exposed 

for 4 h to a range of concentrations from 0.0005 to 10000 μg/mL. The cells were then washed,

resuspended in growth medium, and incubated at 37 ±1 °C for 48 h. The rate of cell growth was 

determined for each of the treated cultures and compared to the rate of growth of the solvent 

controls. The doses of chemical selected for testing were within the range yielding approximately 

0-90% cytotoxicity. For each assay, there were 2-4 solvent controls, a positive control of ethyl 
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methylsulfonate at 4.7 x 10-6 M (or methyl methanesulfonate at 10-20 μg/mL) for the test without 

metabolic activation, and a positive control of 3-methylcholanthrene at 1.86 x 10-5 M (or 

dimethylbenz[a]-anthracene at 0.5-4 μg/mL) for the test with metabolic activation.

The mutagenicity assay was performed according to the procedure described by Clive and 

Spector. A total of 1.2 x 107 cells in duplicate cultures were exposed to the test chemical, positive

control, and solvent control for 4 h at 37 ± 1 °C, washed twice with growth medium, and 

maintained at 37 ±1 °C for 48 h in log-phase growth to allow recovery and mutant expression. 

Cells in the cultures were adjusted to 3 x 105/mL at 24 h intervals. They were then cloned (1 x

106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft 

agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% 

pluronic F-68, and 0.23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to 

trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 μg/mL) to

the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at 

-70 °C and was thawed immediately before use. Plates were incubated at 37 ±1 °C in 5% CO2 in 

air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, 

DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than 

x0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106

surviving cells. Although there are several different methods for evaluating mouse lymphoma 

data, results from this study were interpreted using a doubling of the mutant frequency

over the concurrent solvent-treated control value as an indication of a positive effect, together 

with evidence of a dose-related increase. Doubling of the mutant frequency was previously 

reported as representing a positive effect. Only doses yielding total growth values of 10% were 

used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth 

were used in determining dose response.

The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony 

counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step 

sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in 

colony diameter. The size range used was from 0.2 to 1.1 mm.

RESULTS

Genotoxicity result: Positive

Results remarks: In the nonactivated test, the old evaluation was Positive at a concentration of 

750 μg/mL, and the new evaluation was inconclusive. In the activated test, the old evaluation 

was positive at 25 μg/mL, and the new evaluation was positive at 50 μg/mL.

CONCLUSIONS

Positive in the presence of metabolic activation. Inconclusive without metabolic activation.

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions
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REFERENCE

Seifried et al. 2006. Compilation of two decades of mutagenicity test results with the Ames 

Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays. Chem. Res. 

Toxicol. 19: 627-644.
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B-9. GENETIC TOXICITY – IN VITRO UNSCHEDULED DNA SYNTHESIS 

Study 1: In vitro unscheduled DNA synthesis assay in human embryonic lung fibroblasts 

Title: Determination of Mutagenic/Carcinogenic and Cytotoxic Potential of Four Chemical 

Compounds

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks: Code Identification – TT-100; Sample number – X18BT7565 Cobratec 78669A; 

Description – Tan round crystals

METHOD

Method/guideline followed: Not stated

Test type: Unscheduled DNA Synthesis Assay

GLP compliant?: Not specified 

Year study performed: ~1978

Test species/strain: WI-38 Human Embryonic Lung Fibroblasts

Metabolic activation: None

Concentrations tested: Not legible

Statistical methods used: Not stated

Test method/conditions remarks:

This test system is utilized to determine the potential of compounds to damage DNA in 

mammalian cells.

One mL of the cell suspension (2 x 105 cells/mL) is added to four mL of media in 60-mm tissue

culture plates (6 x replicates per dose level). The cells are allowed to grow to confluency (5-7 

days) then exposed to 10 mM hydroxyurea for 2 hours. The cells are then washed and exposed 

to a mixture of the test material and tritiated thymidine (3

H-TdR) for 24 hours. After the 

exposure the plates are washed 3 X with phosphate buffered saline (pH 7.2) and lysed with 2 mL 

of 0.1M NaOH. After neutralization with 0.2 mL of 1M HCl, 2.2 mL of cold 10 percent TCA is 

added, and the plates incubated at 4 oC for 1 hour. The lysate is then filtered through a GF/A 

filter which is subsequently washed 3 X with TCA. The filters are dried for 8 hours and counted 

in a Nuclear Chicago Mark III liquid scintillation counter.
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Dosage levels which have caused DNA damage will exhibit elevated counts due to the increased 

update of 3H-TdR are mediated by the DNA repair mechanism in the cells.

RESULTS

Genotoxicity result: Negative

Results remarks: The potential for damaging cellular DNA was monitored in WI-38 human 

embryonic lung fibroblasts by measuring the amount of 3H-TdR incorporated into cells exposed 

to subtoxic levels of the chemical samples.

Samples of TT-100 did not elicit significant incorporation above control levels at any of the dose 

levels tested.

CONCLUSIONS

Samples of TT-100 did not elicit significant incorporation above control levels at any of the dose 

levels tested.

STUDY RELIABILITY

Klimisch code = [2] – Valid with restrictions

REFERENCE

TSCATS submission (OTS0516797, Doc ID 86-890000263, received 06/05/89): Huntingdon 

Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 

19 Chemicals with Cover Letter Date 060289. Pp. 72-91.
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B-10. GENETIC TOXICITY – IN VITRO MOUSE FIBROBLAST 

TRANSFORMATION STUDY

Study 1: In vitro mouse fibroblast transformation study

Title: Determination of Mutagenic/Carcinogenic and Cytotoxic Potential of Four Chemical 

Compounds

TEST SUBSTANCE 

Identity: Tolyltriazole, CAS No. 29385-43-1

Remarks:

Code Identification – TT-100

Sample number – X18BT7565 Cobratec 78669A

Description – Tan round crystals

METHOD

Method/guideline followed: Not stated

Test type: C3H10T1/2 Mouse Fibroblast Transformation Assay

GLP compliant?: Not specified 

Year study performed: ~1978

Test species: C3H10T1/2 cell line

Metabolic activation: None

Concentrations tested: 6, 60 and 600 μg

Statistical methods used: Not stated

Test method/conditions remarks: The C3H10T1/2 line is a fibroblastic cell line, derived from 

C3H mouse embryos which are highly sensitive to postconfluence inhibition of cell division. 

When C3H10T1/2 cells are exposed to a variety of chemical carcinogens morphologically 

transformed foci develop with a frequency that is dose dependent. The foci have been shown to 

produce fibrosarcomas when injected into syngeneic mice. Therefore, the C3H10T1/2 cell line 

can be used to assay for chemical carcinogens.

Two sets of 60-mm culture dishes are seeded with cells. One set of dishes, 12 x replicates per 

dose, is seeded with 1000 cells. This set is used for the transformation assay. The second set 

was seeded with 200 cells per dish, 3 x per dose, and is utilized for cytotoxicity determinations.
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Both sets of cells are treated in an identical manner with solutions of the test material. Following 

a 24-hour exposure period, the toxicant is washed from all the plates and the cells refed with 

normal growth media. Ten days following the initial seeding, the plates designated for the 

cytotoxicity assay are washed, fixed with methanol, and stained. The clones on all of the plates 

are counted and the plating efficiency determined. This plating efficiency is expressed as the 

number of colonies formed as a percentage of the number of cells seeded.

The transformation set are incubated for 6 weeks with weekly medium changes. During this 

time, the clones grow to form a confluent monolayer of cells on the bottom of the plates. At the 

termination of the incubation period, all remaining plates are washed, fixed, stained and scored 

for the transformed foci:

Type I Foci: A Type I focus is composed of tightly packed cells 

Type II Foci: The cells display massive piling into opaque multilayers.

Type III Foci: These foci are composed of highly polar, densely stained cells, which form a crisscrossed multilayer.

Type II and Type III foci are considered to be malignantly transformed.

The transformation frequency is calculated as the percentage of cells that give rise to transformed 

foci corrected for plating efficiency which is determined by the cytotoxicity assay.

RESULTS

Endpoint value(s): For the 6, 60, 600 μg and control groups, the transformation frequency was 

0.32, 0.15, 0.07 and 0.07, respectively.

Results remarks: None of the sample chemicals at the dose levels tested produced significant 

increases in transformation above control cell population levels. As a positive control the cells 

did respond in a dose-dependent manner, to the chemical carcinogen dimethylbenzanthracene 

(DMBA).

CONCLUSIONS

None specified

STUDY RELIABILITY

Klimisch code = [2] – Reliable with restrictions. This test represents a non-standard testing 

method.

REFERENCE

TSCATS submission (OTS0516797, Doc ID 86-890000263, received 06/05/89): Huntingdon 

Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 

19 Chemicals with Cover Letter Date 060289, pp. 72-91.
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B-11. CAS No. 29385-43-1: Summary of Endpoints

Endpoint Study Information 

Available

Acceptable Data Gap

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 No - Yes

Gene Mutation Yes Yes No

Chromosomal Aberrations No - Yes

Reproductive Toxicity No - Yes

Developmental Toxicity No - Yes