SRC TR

- 10

- 024

HPV4 CHEMICAL DATA ADEQUA CY 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 Assi gnment Manager David Brooks, Alternate Work Assignment Manager

February 9

, 2010HPV4 Chemical Data Adequacy Review

1H

- Benzotriazole, 4(or 5)

- methyl

- , CAS No. 29385

- 43

- 1

February 2010

DRAFT

Submitted to: High Production Volume Chemicals Bra nch

Risk Assessment Division

Office of Pollution Prevention and Toxic s Environmental Protection Agency

WAM: Meena Sonawane

Alternate WAM: David Brooks

1200 Pe nnsylvania Avenue, NW

Washington, DC 20460

- 0001

Prepared By:

SRC , Inc.

Environmental Science Center 2451 Crystal Drive, Suite 804 Arlington, VA 22202Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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1 A. PREPARATION OF THE DATA ADEQUACY ASSESSMENTS

A

- 1. Source material used for assessment

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

C hromosomal 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 toxic ity data:

PubMed

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

sciences with a concentration on biomedicine

T OXNET

- N ational L ibrary of M edicine databases on toxicology, hazardous chemicals, environmental health, and toxic releases; includes:

**•** TOXLINE with BIOSIS and NTIS ;

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

**•** Chemical Carcinogenesis Research Information System (CCRIS)

- Carcinogenicity and mutagenicity test results for over 8000

**•** chemicals) ;

**•** Hazardo us Substances Data Bank (HSDB)

- Comprehensive, peer

reviewed toxicology data for about 5000 chemicals ;

**•** Genetic Toxicology Data Bank (GENE

- TOX)

- Peer

- reviewed genetic toxicology test data for over 3000 chemicals.

TSCATS

- U npublished technical reports su bmitted by industry to the EPA under the

Toxic Substances Control Act

Registry of toxic effects of chemical substances (RTECs)

Environmental Fate Database (EFDB)

ECOTOX

- EPA’s peer

- reviewed ecotoxicology databaseDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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2 EPA’s Integrated Risk Information Syst em (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 INC HE M or CICADs)

European Chemical Substances Information System (ESIS)

- source of IUCLID Datasets

Any available IUCLID Datasets found through the ESIS website and robust summaries submitted to EPA by former sponsors on the assigned chemicals were retrieved. Original

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

While IUCLID D ataset s sometimes contained studies of interest, certain study descriptions contained insufficient detail to assess adequacy. In these cases, the primary source

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

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 a dequacy. 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 com bined 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 tab le 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 f ollowing pages. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint is determined to be inadequate, a basis for that conclusion is supplied. NOTE: If no relevant studies were identified for the chemical , only the data availability/adequacy summary table was completed for the report

.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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3 B

- 1. ACUTE TOXICITY TO FISH

Summary of endpoint: A dequate

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

Iden tity: 5

- Methyl

- 1H

- Benzotriazole or 4

- Methyl

- 1H

- Benzotriazole (MeBT), CAS No. 29385

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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 LC 50 ; static

- renewal renewed at 50 hours with freshly

- prep ared 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. Enviro nmental 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. LC 50 values were calculatedDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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4 using either the graphi cal, probit, Spearman

- Karber, or trimmed Spearman

- Karber methods according to U.S. Environmental Protection Agency established criteria.

RESULTS

Endpoint value(s): The 96

- hour LC 50 v alue for fathead minnows was 38

- 65 mg/L.

Results remarks: Measured hig h 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 LC 50 was calculated based

on the measured 10 0% 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. 2 9385

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5 Study 2: Acute toxicity study in fathead minnows

Title: The Acute Toxicity of Tolyltriazole

- Cobrat ec TT

- 100 Sample 1090 to the Fathead

Minnow Pimephales promelas

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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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 LC 50

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 w ere 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 reconstitute d distilled water.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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6 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’D DT 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 serv ed 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 usuall y preceded by loss of equilibrium.

Litchfield and Wilcoxon’s method was used to compute the confidence limits for the LC1, LC 50 , and LC99 (at 24, 48 and 96 hours).

RESULTS

Endpoint value(s):

24 hour:

LC 1 = 23.00 ppm

LC 50 = 36.90 ppm

LC 99 = 68.50 ppm

4 8 hour:

LC 1 = 14.00 ppm

LC 50 = 29.80 ppm

LC 99 = 62.50 ppmDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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7 96 hour:

LC 1 = 14.20 ppm

LC 50 = 25.50 ppm

LC 99 = 46.00 ppm

Results remarks:

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

24 hour:

LC 1 = 0.0460 ppm

LC 50 = 0.1180 ppm LC 99 = 0.3010 ppm

48 hour:

LC 1 = 0.0485 ppm LC 50 = 0.1010 ppm LC 99 = 0.2110 ppm

96 hour:

LC 1 = 0.0360 ppm LC 50 = 0.0945 ppm LC 99 = 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 RELIABILIT Y

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

- Cob ratec 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. 2 9385

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8 Study 3: Acute toxicity study in bluegill sunfish

Title: The Acute Toxicity of Tolyltriazole

- Cobratec TT

- 100 Sample 1090 to the Bluegi ll Sunfish ( Lepomis macrochirus )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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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 LC 50

GLP com pliant?: 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 wate r.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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9 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, a cetone (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 partic ular 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 h ours 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 LC 1 , LC 50 , and LC 99 (at 24, 48 and 96 hours).

RESULTS

Endpoint value(s):

24 hour:

LC 1 = 3.90 ppm LC 50 = 4.02 ppm LC 99 = 4.81 ppm

48 hour:

LC 1 = 2.55 ppm LC 50 = 3.55 p pm LC 99 = 4.90 ppmDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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10 96 hour:

LC 1 = 2.30 ppm LC 50 = 3.47 ppm LC 99 = 5.20 ppm

Results remarks:

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

24 hour:

LC 1 = 0.0084 ppm LC 50 = 0.0126 ppm LC 99 = 0.0192 ppm

48 hour:

LC 1 = 0.0063 ppm LC 50 = 0.01 10 ppm LC 99 = 0.0192 ppm

96 hour:

LC 1 = 0.0065 ppm LC 50 = 0.0106 ppm LC 99 = 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 res trictions.

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. 2 9385

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11 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 )

Thi s study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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Remarks: Referred to as Tolyltriazole

- Cobratec TT

- 100, Sample 1090. Supplied as tan granules.

METHOD

Method/guidel ine 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 LC 50

GLP compliant?: Not indicated

Year study wa s 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. 2 9385

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12 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 c ontrols.

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 LC 50 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 specifiedDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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13 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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14 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

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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 LC 50

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 hel d 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 rec onstituted distilled water. Water Temperature: 52 ±2 oF.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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15 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, 0.031 6, 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 se rved 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 usual ly 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 LC 50 is determined to be 21.40 ppm with 95% confidence limits of 18.94 to 24.18

ppm.

Res ults remarks:

CONCLUSIONS

None specifiedDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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16 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. 2 9385

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17 Study 6: Acute toxicity study in fathead minnows

Title: A 96

- Hour Static

- Rene wal Acute Toxicity Test with the Fathead Minnow (Pimephales promelas)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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Remarks: Off

- white particles

METHOD

Metho d/guideline followed: OECD Guideline 203

Test type: Acute LC 50 ; 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; stand ard 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 consultat ion 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 di stributed 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 LC 50 values and 95% confidence limits at 24, 48, 72 and 96 hours. The no mortality concentration was determined by visually exami ning the mortality data.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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18 RESULTS

Endpoint value(s): The 96

- hour LC 50 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 CaCO 3 , and 144 mg/L as CaCO 3 , respectively. At test termination (96 hours) they were 325 umhos/cm, 184 mg/L as CaCO 3 , and 144 mg/L as CaCO 3 , respectively, in the Negative Control Replicate A.

After the test substance was added to the vortex, a clear colorles s 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.

F ish 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 wi th 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 LC 50 value for fathead minnows exposed to test substance was 43 mg/L. The lower and upper 95% confid ence 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. 2 9385

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19 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

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- 1)

Remarks: R eferred 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 LC 50

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 Continent al 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 CaCO 3 , total alkalinity of 30 mg/L as CaCO 3 and specific conductance of 1 55 μ mhos/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 Met er.

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 dir ectly to the test containers.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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20 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

rand om 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, di ssolved 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. LC 50 s could not be calculated for the 24 and 48 hour exposure periods due to the lack

of sufficient mortalities. LC 50 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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21 Study 8: Acute toxicity study in rainbow trout

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

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 amb er, w ater

- soluble liquid.

METHOD

Method/guideline followed: Committee on Methods for Toxicity Tests with Aquatic Organisms (1975)

Test type: Acute LC 50

GLP compliant?: Not specified

Year study was performed: 1979

Test species: Rainbow trout ( Salmo g airdneri ) 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: Di lution 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, tota l hardness of 42 mg/L as CaCO 3 , total alkalinity of 28 mg/L as CaCO 3 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

ORIO N 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 concentrati ons 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 19.6Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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22 liter, chemically clean glass jars, each c ontaining 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 nom inal 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 l ethal 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. LC 50 calculations are based on nominal

concentrat ions of the test material in soft reconstituted water.

RESULTS

Endpoint value(s): The 96

- hour LC 50 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 , rece ived 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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23 B

- 2. ACUTE TOXICITY TO DAPHNIA

Summary of en dpoint: 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

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Remarks: None

METHOD

Method/guideline followed: U.S. Environmental Protection Agency. 1985. Methods for measuring the acute toxicity of effluents and receiv ing waters to freshwater and marine microorganisms. EPA 600/4

- 85

- 013. Environmental Monitoring and Support Laboratory, Cincinnati, OH.

Test type: Acute EC 50 ; Static

GLP compliant?: Not specified

Year study was performed: ~1999

Test species: Water fl eas ( 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 LC 50 values were calculated using either the graphical,Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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24 probit, Spearman

- Karber, or trimmed Spearman

- Karber methods according to U.S. Environmental Protection Agency

- established criteria.

RESULTS

Endpoint value(s): The 48

- hour EC 50 value for C. dubia was 1 02

- 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 L C 50 s 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] Reliabl e 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. 2 9385

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25 Study 2: Acute toxicity study in Daphnia magna

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

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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Remarks: Referred to as RM 27513 (EnviroSystems Sampl e 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 p rocedures 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 wa s 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 a t 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 theDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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26 temperature was 19.0

– 19.8 oC. Daphnids were fed yeast, trout chow, an d/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

En viroSystems 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 te mperature 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 provid ed 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 immo bilization (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 a fter 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 interprete d by standard statistical techniques (Stephan, 1983). Nominal concentrations of test substance the number of daphnids killed or immobilized were

used to calculate the EC 50 value.

RESULTS

Endpoint value(s): Exposure of daphnids to the test substance resu lted in a 48

- hour EC 50 of 19 mg/L Rm27513, with a 95% confidence interval of 12

– 25 mg/L. 24

- hour EC 50 = 34 mg/L

Results remarks: Test vessels containing RM 27513 appeared clear initially and remained free of insoluble material throughout the test. Nin ety

- 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 toDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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27 the referen ce toxicant sodium dodecyl sulfate resulted in a 48 hour median lethal concentration (LC 50 ) 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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28 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

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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 s tatic

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 s terile enriched media identical to media used for this test and

maintained at test conditions.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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29 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 conduc ted 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 ve ssels 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 ph otoperiod was maintained with

cool

- white fluorescent lights that provided a light intensity of 35 uEs - 1 m - 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 we re interpreted by standard statistical techniques (Stephan, 1983). The binomial or moving average method was used by the author to

calculate EC 50 values using nominal concentrations of test substance. The NOEC was calculated using Dunnett’s test, which i ncludes 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 b y 100. The percent change from control was used to compute EC 50 and NOEC values.

RESULTS

Endpoint value(s): The 72

- hour EC 50 is <62.5 mg/L, and the 72

- hour NOEC is 250 mg/L RM

27513. The 96

- hour EC 50 is 87 mg/L and the 96

- hour NOEC is 250 mg/L.

24

- hou r EC 50 > 1000 mg/L

48

- hour EC 50 = 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 restrictionsDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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30 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

- 44Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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31 B

- 4

. ACUTE TOXICITY

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats

Title: Acute Oral LD 50 Investigation in Rats of Cobratec TT 100 Sample No. 5459

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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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

Stati stical methods used: An LD 50 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): LD 50 = 675 mg/kg (95% confidence limits were 535

- 851 mg/kg)Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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32 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 d ay 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 ): Hunting don Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 19 Chemicals with Cover Letter Date 060289. pp. 32

- 34.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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33 Study 2: Acute oral toxicity study in rats

Title: The Acute Oral Toxicity ( LD 50 ) to Rats of Ol in 58734

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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

METHOD

Method/guideline followed: Me thod 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 ani mals/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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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34 RESULTS

Endpoint value(s): LD 50 = 1830 mg/kg

- bw

Results remarks: Deaths occurred in 3 hours to 5 minutes, following CNS depression, shallow breathing, lacri mation, 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 LD 50 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; lack s 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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35 Study 3 : Acute in halation 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

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Remarks: Ref erred to as Cobratec RTT 100, Sample No. 3000. The compound was a fine powder, off

- white in color. The melting p oint 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 p erformed:

~ 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

Statisti cal 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 wa s 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 followin g 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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36 Animals were allowed 7 days to accommodat e 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 o C 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 atm osphere. 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): LC 50 > 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 moderateDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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37 incidence of hemorrhage or hepatization, was found in all animals. Some incidence of pale liver and kidneys was also noted in males and f emales.

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 su bmission: Evaluation of the Acute Inhalation Toxicity of Tolyltriazole (Final Report) with Attachments and Cover Letter Dated

061289.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

38 Study 4 : Acute dermal toxicity study in rabbits

Title: Report on Acute Dermal Toxicity Investigation in Rabbits of Cob ratec 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 ani mals/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

rab bits 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): LD 50 > 2000 mg/kgDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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39 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 obser vation. No additional signs of skin irritation were observed.

CONCLUSIONS

None stated

STUDY RELIABILITY

Klim i sch code = [2]

– Valid with restrictions

REFERENCE

TSCATS submission ( OTS0516797 , Doc ID 86

- 890000263 , received 06/05/89 ): Huntingdon

Resear ch Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for 19 Chemicals with Cover Letter Date 060289. pp. 10

- 15.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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40 Study 5 : Acute dermal toxicity study in rabbits

Title: The Acute Dermal Toxicity to Rabbits of Olin 5873 4

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/se x/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 leve ls 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 c ontinuous 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 periodDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

41 RESULTS

Endpoint value(s): LD 50 > 4000 mg/kg

- bw

Results remarks: No death s 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 i s greater than 4 g/kg

- bw and this product

evokes no irritation.

STUDY RELIABILITY

Klimisch code = [3]

– Lack of study details; study is out dated

REFERENCE

TSCATS submission (OTS0520643

, Doc ID 86

- 89000060A , received 06/13/89 ): Huntingdon

Research Cente r, 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

42 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 a nd 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 b inder.

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

Su bstances Act.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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43 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 Studie s for 19 Chemicals with Cover Letter Date 060289. pp. 51

- 57.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

44 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. B oth 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 drop ped. 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 reacti ons 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 ultra

violet lamp to detect corneal damage if present.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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45 The reactions were scored from: “Illu strated 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

irrit ation 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 treatment

related 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, detail s 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

– sw elling 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 sub mission (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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

46 Study 2: Eye irritation in rabbits study

Ti tle: 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: M ethod 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

wash out.

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 Regulation s

RESULTS

Endpoint value(s): Slightly to transiently irritatingDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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47 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 da y 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

Klimis ch 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

48 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 SUBSTANC E

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 T h., 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 pi g 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

Rou te 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 “AppraisalDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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49 of the Safety of Chemicals in Foods, Drugs and Cosmetics” (1959), the US Association of Food and Drug Officials (AFDO).

The test was performe d 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. A n 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

weekend s) 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 th e 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”).Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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50 Ten days after the intrac utaneous 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

band ages 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.

CONC LUSIONS

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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

51 B

- 8

. GENE MUTATION

Summary of endpoint: Adequate

Study 1: In vitro m utation in Salmonella study

Title: A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mous e 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., McCan n, 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 Oxoi d nutrient broth no. 2 and were removed from incub ation 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 wa s 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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52 S9 Preparation. Liver S9 ho mogenate 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 compon ents of the S9 mix were 8 mM MgCl 2 , 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 wa s added.

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

strain and 50 μ L 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 μ L of tester strain, and 50 μ L 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 p lates 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 plat e 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 incr easing

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 cont aining 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 statedDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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53 Results remarks: N one 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 l ymphoma cell mutation assays. Chem. Res.

Toxicol. 19: 627

- 644.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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54 Study 2: In vitro m utation 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

– Salmonel la 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 br oth, 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 use d. 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 co ntents of the tubes were mixed and poured onto the surface of petriDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

- 1

55 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.

Pla tes 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, TA 98, 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 negativ e 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 th ese 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 TA 97 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 mutageni city 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. Occasion ally, 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 dose

related 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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56 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 651 117. 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

- 158Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

- 43

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57 Study 3: In vitro mutation in Salmonella st udy

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: Salmonel la 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 10 8 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 the n 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)Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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58 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 additiona l 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 i s 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

TA15 35 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 wit h 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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59 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

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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: 19 82

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

Metabolic activation: With and without

Concentrations tested: 25, 75, 67 5, 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 contro ls. 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: N

methyl

- N’

- nitro

- N

- nitrosoguanidine, 3 and 5 μg/0.1 mL phosphate buffer; 4) for strain TA1537: 9(5)aminoacridine hydrochloride monohydrate, 50 an d 100 μg/0.1 mL DMSO; 5) for strainDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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60 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 res ult: 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

- prototrop hic 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 abo ve.

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

– Micro some Mutagenicity Test with

TK 10 637/1 with Cover Letter Dated 060589.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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61 Study 5: In vitro mo use 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

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Remarks: None

METHOD

Method/guideline followed: Not specified

Test type: L5178Y Mouse Lymph oma 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 screen ed for the presence of mycoplasma after cryopreservation.

New cultures were initiated at approximately 3 mo nth 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 10 5 /mL (6 x 10 6 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 μ g/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 wi thin the range yielding approximately

0

- 90% cytotoxicity. For each assay, there were 2

- 4 solvent controls, a positive control of ethylDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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62 methylsulfonate at 4.7 x 10 - 6 M (or methyl methanesulfonate at 10

- 20 μ g/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 μ g/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 10 7 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 i n log

- phase growth to allow recovery and mutant expression.

Cells in the cultures were adjusted to 3 x 10 5 /mL at 24 h intervals. They were then cloned (1 x 10 6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft

ag ar 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 μ g/mL) t o the cloning medium for mutant selection. The 100 x 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% CO 2 in

air for 10

- 12 days and then counted with an Artek automated colon y counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than

x 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 10 6
surviving cells. Although there are several different me thods 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 in crease. 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 coloni es 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 with out metabolic activation.

STUDY RELIABILITY

Klimisch code = [2]

– Valid with restrictionsDraft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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63 REFERENCE

Seifried et al. 2006. Compilation of two decades of mutagenicity test results with the Ames Salmonella typhimurium and L5178Y mouse lymphoma cell muta tion assays. Chem. Res.

Toxicol. 19: 627

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64 B

- 9 . GENETIC TOXICITY

– IN VITRO UNSCHEDULED DNA SYNTHESIS

Study 1: In vitro un scheduled DNA sy nthesis assay in human embryonic lung fibroblasts

Title: Determination of Mutagenic/Carcinogenic and Cytot oxic Potential of Four Chemical Compounds

TEST SUBSTANCE

Identity: Tolyltriazole, CAS No. 29385

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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 10 5 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 fo r 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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65 Dosage levels which have caused DNA damage w ill exhibit elevated counts due to the increased update of 3 H

- 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 embryoni c lung fibroblasts by measuring the amount of 3 H

- 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 0 6/05/89 ): Huntingdon Research Center, Union Carbide Corp. 1989. Initial submission: Health and Safety Studies for

19 Chemicals w ith Cover Letter Date 060289. Pp. 72

- 91

.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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66 B

- 10 . GENETIC TOXICITY

– IN VITRO MOUSE FIBROBLAST

TRANSFORMATION STUDY

Stud y 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

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- 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 highl y 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 wi th 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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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67 Both sets of cells are treated in an identical manner with solutions of the t est 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 me thanol, 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 incub ated 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 cris s

crossed 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 signi ficant 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

Kli misch 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 submissi on: Health and Safety Studies for

19 Chemical s with Cover Letter Date 060289, pp. 72

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.Draft HPV4 Chemical Data Adequacy Review CAS No. 2 9385

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68 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

Ac ute 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 Developmenta l Toxicity No

- Yes