SRC TR-12-037

HPV2 TEST RULE DATA ADEQUACY REVIEW: MAY 2012 UPDATE (FINAL)

Benzenediamine, ar, ar-diethyl-ar-methyl-, CASRN 68479-98-1

Prepared by:

Jennifer Rayner, William L. Richards, Teresa Manyin, and David Wohlers

Chemical, Biological and Environmental Center

SRC, Inc.

7502 Round Pond Road

North Syracuse, NY 13212

Contract No. EP-W-09-027

Task FG007.2.009.001

Submitted to:

OCSPP/OPPT/RAD (7403M)

U.S. Environmental Protection Agency

1200 Pennsylvania Avenue

Washington, D.C. 20460

Debra Milligan, Project Officer

Meena Sonawane, Work Assignment Manager

David Brooks, Alternate Work Assignment Manager

May 4, 2012
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TABLE OF CONTENTS

A. INTRODUCTION .................................................................................................................... 1

B. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE ........ 1

C. DATA FOR SIDS ENDPOINTS.............................................................................................. 2

C-1. Acute Toxicity to Fish: Test Requirement (C1) under Final HPV2 Test Rule Dated 

January 7, 2011 ........................................................................................................................... 2

C-2. Acute Toxicity to Daphnia: Test Requirement (C1) under Final HPV2 Test Rule Dated 

January 7, 2011 ........................................................................................................................... 3

C-3. Toxicity to Plants (Algae): Test Requirement (C1) under Final HPV2 Test Rule Dated 

January 7, 2011 ........................................................................................................................... 4

C-4. Acute Toxicity.................................................................................................................... 8

C-5. Repeated-Dose Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011 ......................................................................................................................... 14

C-6. Genetic Toxicity – Gene Mutation: Test Requirement (E1) under Final HPV2 Test Rule 

Dated January 7, 2011............................................................................................................... 33

C-7. Genetic Toxicity – Chromosomal Aberrations: Test Requirement (E2) under Final 

HPV2 Test Rule Dated January 7, 2011 ................................................................................. 111

C-8. Reproductive Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011 ....................................................................................................................... 121

C-9. Developmental Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011 ....................................................................................................................... 122

D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)...................................................... 123

D-1. Genetic Toxicity – Other................................................................................................ 123

D-2. Carcinogenicity .............................................................................................................. 150

D-3. Skin Irritation ................................................................................................................. 164

D-4. Eye Irritation .................................................................................................................. 166

D-5. Skin Sensitization........................................................................................................... 168

D-6. Toxicokinetic Studies..................................................................................................... 170

E. DATA ADEQUACY SUMMARY TABLE......................................................................... 179
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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A. INTRODUCTION

This document evaluates a submission from Albemarle Corporation and Lonza (dated March 1, 

2012) received by EPA in response to the final test rule, “Testing of Certain High Production 

Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to 

herein as HPV2) for benzenediamine, ar, ar-diethyl-ar-methyl- (CASRN 68479-98-1). In this 

submission, Albemarle Corporation and Lonza supplied data for genetic toxicity tests required

by the HPV2 test rule: chromosomal aberrations and mammalian cell mutation. These newly 

submitted studies are evaluated for adequacy herein. This report also contains data, evaluated 

previously by EPA, that were derived from a prior submission by Albemarle and Lonza 

(received by EPA on May 6, 2011) and, therefore, provides an update of the data adequacy 

assessment previously compiled for CASRN 68479-98-1 in August 2011.

Data available for SIDS toxicity endpoints are evaluated for adequacy in Section C. Using 

standard testing guidelines and professional judgment as guides, each study was assigned a 

Klimisch reliability code. Additional data for non-SIDS toxicity endpoints (“supplemental” 

data) are reviewed in Section D. A summary of the adequacy assessment for each SIDS 

endpoint is provided on the last page of this document (Section E).

B. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

The previous data adequacy assessment (dated August 2011) for CASRN 68479-98-1 identified 

the following data gaps for endpoints required by the HPV2 test rule: acute toxicity to fish, acute 

toxicity to Daphnia, chromosomal aberrations, and reproductive/developmental toxicity. The 

test rule requirement for chromosomal aberrations (E2) may be satisfied by conducting one of 

the following tests: (1) In vitro Mammalian Chromosome Aberration Test (40 CFR 799.9537), 

(2) Mammalian Bone Marrow Chromosomal Aberration Test (in vivo in rodents: mouse 

[preferred species], rat, or Chinese hamster; 40 CFR 799.9538), or (3) Mammalian Erythrocyte 

Micronucleus Test (sampled in bone marrow; in vivo in rodents: mouse [preferred species], rat, 

or Chinese hamster; 40 CFR 799.9539). 

Genotoxicity data supplied on March 1, 2012 by Albemarle Corporation and Lonza were 

adequate to satisfy the chromosomal aberrations (E2) test requirement promulgated in the final 

HPV2 test rule. These newly submitted data included a chromosomal aberration study in human 

lymphocytes (see p. 111 of this report; Study 1 in section C-7) and a bone marrow micronucleus 

test in mice (see p. 115 of this report; Study 2 in section C-7). No additional studies need to be 

conducted for the E2 endpoint. Additional genotoxicity data, included in the March 1, 2012 

submission from Albemarle Corporation and Lonza, consisted of an in vitro gene mutation test in 

mammalian cells, which provided further support for the gene mutation endpoint (see p. 72 of 

this report; Study 12 in section C-6).

The following data gaps remain for test rule-required endpoints: acute toxicity to fish, acute 

toxicity to Daphnia, and reproductive/developmental toxicity.
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C. DATA FOR SIDS ENDPOINTS

C-1. Acute Toxicity to Fish: Test Requirement (C1) under Final HPV2 Test Rule Dated 

January 7, 2011

Summary of endpoint: Not adequate

No studies were submitted for this endpoint.
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C-2. Acute Toxicity to Daphnia: Test Requirement (C1) under Final HPV2 Test Rule 

Dated January 7, 2011

Summary of endpoint: Not adequate

No studies were submitted for this endpoint.
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C-3. Toxicity to Plants (Algae): Test Requirement (C1) under Final HPV2 Test Rule 

Dated January 7, 2011

Summary of endpoint: Adequate

Study 1: Toxicity to Desmodesmus subspicatus (Caspers, 2010)

Title: Alga, growth inhibition test with DETDA (Diethyltoluylendiamin-80).

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Physical state: clear, light-colored liquid; Test item: DETDA 

(Diethyltoluylendiamin-80); Chemical name: Diethylmethylbenzoldiamine; CAS Number: 

68479-98-1); Purity: 97.7%; Composition: 2,4-DETDA (75-81%), 2,6-DETDA (18-24%), 2,4,6-

TEMPDA (0-1%)

METHOD

Method/guideline followed: OECD Guideline 201 (2006); EU method C.3 (2009)

Test type: Toxicity test to freshwater algae

GLP compliant?: Yes

Year study was performed: 2010

Test species: Freshwater green algae (Desmodesmus subspicatus, formerly Scenedesmus 

subspicatus) 

Analytical monitoring: Yes

Exposure duration: 72 hours

Concentrations tested: Nominal: 0.1, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L Measured (GC with 

FID detector) concentrations ranged from 95.3 to 102.2% of nominal values at initiation and 83.1 

to 95.5% of nominal at 72 hours. 

Test method/conditions remarks: Endpoints were growth rate and biomass. Exponentially 

growing algae cells were exposed for 72 hours to a range of concentrations; nominally 0.1, 0.32, 

1.0, 3.2, 10, 32, and 100 mg/L of DETDA dissolved in dilution water. Measured (GC with FID 

detector) concentrations ranged from 95.3 to 102.2% of nominal values at initiation and 83.1 to 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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95.5% of nominal at 72 hours. The cell densities were measured at 24-hour intervals. Inhibition 

of algal population was measured as reduction in growth rate relative to control cultures. The 

72- hour EC10 and EC50 were calculated by probit analysis. All calculations were carried out 

using the statistics program ToxRatPro Version 2.10 (released 2010-02-20). For the calculations 

all algae counts were divided by a factor of 10,000. NOEC and LOEC were determined by a 

multisampling comparison.

During the test a temperature range of 21-24°C was maintained in the test vessels. The pH was 

measured at the beginning of the test and after 72 hours of exposure. The maintenance of the test 

item concentrations was proved by analytical measurements. In order to avoid an impairment of 

the test system, an additional replicate was used for analysis and pH measurement at the 

beginning of the test. Chemical analysis and pH measurements at the end of the test were 

performed using replicate I of the test concentrations and control vessels, respectively. 

Test Species

Name: Desmodesmus subspicatus Strain No. 86.81 SAG

Source: Strain of the test species obtained from ‘The Collection of Algal Cultures’ of the 

Institute of Plant Physiology at the University of Gottingen (Germany).

Maintenance and Acclimatization: Exponentially growing stock cultures were maintained in the 

test facility under constant temperature conditions (21-24°C with a maximum fluctuation of +/-

2°C) at a light intensity in the range 60 – 120 μE. x m-2 x -1 (measured in the range 400 to 700 

nm using a spherical flux meter). The growth medium (according to BRINGMANN & KUHN 

(1977) was renewed once a week. Cell density measurements were made using a microcell 

counter, Sysmex F300, Digitana.

Preparation of pre-cultures: Pre-cultures were set up three days before the start of a test. They 

were grown under identical exposure conditions as the stock cultures, except from the use of a 

different growth medium.

Test cultures: The algal inocula for a test were taken from an exponentially growing pre-culture 

and were mixed with the growth medium to make up to a final density of about 5000 cells per 

milliliter in the test medium.

Growth medium and dilution water

Growth medium (OECD medium of OECD TG 201) was used for the growth of the algae in the 

pre cultures and the preparation of stock and test solutions of the test item.

Pre-treatment of test item and preparation of test item concentrations 

A stock solution was prepared to give the desired series of test concentrations. 100.4 mg of the 

test item were added to 1 litre of dilution water and treated for 60 seconds at 8000 rpm with an 

ultra turrax and afterwards stirred for 24 h on a magnetic stirrer. The pH was measured to be 8.0. 

To produce the different test item concentrations appropriate amounts of the stock solution were 

diluted with dilution water to a volume of 100 mL. Afterwards 0.724 mL of the algal inoculum 

were added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item 

concentration and the control 3 replicates were prepared. 
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Exposure Conditions

The test vessels were 300 mL Erlenmeyer flasks, and the culturing apparatus included a light 

chamber in which a temperature in the range 21°C to 24°C was maintained at +/- 2°C, and 

continuous uniform illumination was provided in the spectral range 400 to 700 nm. Temperature 

was measured and recorded daily in a water filled flask which was incubated under the same 

conditions as the test flasks. At the average of the test solutions, a light intensity in the range 60 

to 120 μE. x m-2 x s-1, or an equivalent range of 4000 to 8000 lx, was used. Light intensity was 

checked before the start of the study.

Cell densities were measured in a microcell counter by taking small aliquots from each test flask 

for measurements.

The experimental design of the study included seven test concentrations plus one control. Three 

replicates per concentration and three replicates per control were used. The initial cell density in 

the test cultures was approximately 5000 cells per millilitre. The pH and values at the start and 

the end of the study ranged from 7.9-10.4. Water hardness of the final nutrient medium was 22.5 

mg/L CaCO3.

RESULTS

Endpoint value(s): Based on nominal concentrations reported: ErC50 = 104 mg/L; ErC10 = 54 

mg/L; NOEC (growth rate) = 32 mg/L; LOEC (growth rate) = 100 mg/L; EyC50 = 60 mg/L; 

EyC10 = 37 mg/L; NOEC (yield) = 10 mg/L; LOEC (yield) = 32 mg/L

Results remarks: The following validity criteria of the test were met:

The cell density in the control cultures increased by a factor of at least 16 within 72 hours. The 

test period may be shortened to at least 48 hours to maintain unlimited, exponential growth 

during the test as long as the minimum multiplication factor of 16 is reached. 

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 

2-3, for 72 hour tests) in the control cultures did not exceed 35%. 

The coefficient of variation of average specific growth rates during the whole test period in 

replicate control cultures did not exceed 7%.

CONCLUSIONS

The 72-hr EC50 was found to be 104 mg/L for growth rate and 60 mg/L for yield. The 72-hour 

NOEC and LOEC for growth rate were were 32 and 100 mg/L, respectively. The 72-hour 

NOEC and LOEC for yield were 10 and 32 mg/L, respectively.

STUDY RELIABILITY 

[1] Reliable without restrictions.
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REFERENCE

Caspers. 2010. Alga, growth inhibition with DETDA (Diethyltoluylendiamin-80); Study No. 

2010/0109/01. Performing Laboratory: CURRENTA GmbH & Co. OHG. Leverkusen, 

Germany. Sponsor: Albemarle Corporation, Louvain-la-Neuve, Belgium. 
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C-4. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats (Heenehan et al., 1977)

Title: Acute Oral Toxicity Study

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity of the tested commercial product was not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral -- Rat

GLP compliant?: Not indicated

Year study performed: 1977

Test species: Wistar rats

Number of animals/sex/dose: 5

Route of administration: Oral gavage

Vehicle: Corn oil

Doses/concentrations tested: 280, 400, 560, 780 or 1100 mg/kg

Statistical methods used: None specified

Test method/conditions remarks: Wistar rats (5/sex/dose) were administered a single dose of 

DETDA by gavage in corn oil at doses of 280, 400, 560, 780 or 1100 mg/kg. Animals were 

observed for 14 days.
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RESULTS

Endpoint value(s): LD50 = 485 mg/kg-bw

Results remarks: No additional details were available from this study summary.

CONCLUSIONS

The oral LD50 was estimated to be 485 mg/kg-bw with 95% confidence limits of 406 and 564 

mg/kg-bw.

STUDY RELIABILITY

[2] Reliable with restrictions. Missing details included test method/guideline followed, 

purity/composition of the tested commercial product, clinical observations/ findings, and whether 

or not body weight data were recorded.

REFERENCES

Heenehan, P., Braun, W., and Rinehart, W. 1977. Acute Oral Toxicity Study. Compound: 

Diethylated Toluenediamine. Project No. 4448-77. Performing Laboratory: Bio/dynamics Inc. 

Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 

2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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Study 2: Acute inhalation toxicity study in rats (Thackara and Rinehart, 1978)

Title: An Acute Inhalation Toxicity Study of Diethylated Toluenediamine in the Rat

This study is: Adequate Not Adequate

This study is not adequate for the following reasons: (1) Duration of exposure to the test 

substance was for 1 hour rather than 4 hours as stipulated by the OPPTS guideline 870.1300 for 

Acute Inhalation Toxicity; (2) The concentration of test substance in the breathing zone was not 

measured as stipulated by guideline 870.1300; and (3) The study tested a single concentration 

rather than three as stipulated by guideline 870.1300. Details missing from the study summary 

included purity and composition of the tested commercial product and test method/guideline 

followed.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity of the tested commercial product was not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Acute Inhalation -- Rat

GLP compliant?: Not indicated

Year study performed: 1978

Test species: Sprague-Dawley rats

Number of animals/sex/dose: 5

Route of administration: Inhalation, 1 hr

Vehicle: None

Doses/concentrations tested: 2.45 mg/L

Statistical methods used: None specified
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Test method/conditions remarks: Sprague Dawley rats (5/sex/dose) were exposed for 1 hour 

to a nominal exposure concentration of 2.45 mg DETDA/L. No additional details were available 

from this study summary.

RESULTS

No mortality occurred during the 14 d observation period. Signs of toxicity were observed during 

the exposure and included excessive lacrimation and salivation, mucoid nasal discharge, labored 

breathing, and inactivity. Most of the observed signs disappeared during the 4 hr period postdosing. Lung discoloration was observed at necropsy on d 14. 

Endpoint value(s): LD50 = 2.45 mg/L

Results remarks: No additional details were available from this study summary.

CONCLUSIONS

The inhalation LD50 (1-hr) was > 2.45 mg/L.

STUDY RELIABILITY

[3] Not reliable. Based on information that the sponsor provided in an abstract, this study is not 

reliable for the following reasons: (1) Duration of exposure to the test substance was for 1 hour 

rather than 4 hours as stipulated by the OPPTS guideline 870.1300 for Acute Inhalation Toxicity; 

(2) The concentration of test substance in the breathing zone was not measured as stipulated by 

guideline 870.1300; and (3) The study tested a single concentration rather than three as stipulated 

by guideline 870.1300. Details missing from the study summary included purity and 

composition of the tested commercial product and test method/guideline followed.

REFERENCES

Thackar, J., and Rinehart, W. 1978. An Acute Inhalation Toxicity Study of Diethylated 

Toluenediamine in the Rat. Project No. 77-1962. Performing Laboratory: Bio/dynamics Inc. 

Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 

2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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Study 3: Acute dermal toxicity study in rabbits (Heenehan et al., 1977)

Title: Acute Dermal Toxicity Study

This study is: Adequate Not Adequate

This study is not adequate because important study details were missing from the study summary 

(including purity and composition of the test substance and test method/guideline followed) and

the LD50 could not be determined.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Acute Dermal -- Rabbit

GLP compliant?: Not indicated

Year study performed: 1977

Test species: New Zealand White albino rabbits

Number of animals/sex/dose: 4, sex not indicated

Route of administration: Dermal

Vehicle: None

Doses/concentrations tested: 705, 1000, 1410 and 2000 mg/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: A total of 16 New Zealand White albino rabbits were treated 

dermally with DETDA at doses of 705, 1000, 14 10 and 2000 mg/kg. The hair was clipped prior 

treatment, and the dermal stratum corneum of half the rabbits abraded at the application site. The 

test material was held in place with an impermeable bandage throughout the 24 hr exposure

period. Animals were observed for 14 days.
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RESULTS

There were 1, 4, 2 and 3 deaths at 705, 1000, 1410 and 2000 mg/kg-bw, respectively

Endpoint value(s): LD50 = not determined

Results remarks: No additional details were available from this study summary.

CONCLUSIONS

Due to inconsistent mortality, a meaningful LD50 could not be determined.

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the study summary included purity and 

composition of the test substance and test method/guideline followed. Additionally, the LD50

could not be determined. 

REFERENCES

Heenehan, P., Braun, W., and Rinehart, W. 1977. Acute Dermal Toxicity Study. Compound: 

Diethylated Toluenediamine. Project No. 4449-77. Performing Laboratory: Bio/dynamics Inc. 

Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 

2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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C-5. Repeated-Dose Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011

Summary of endpoint: Adequate

Study 1: 90-Day repeated-dose oral toxicity study in rats (Naismith and Mathews, 1987)

Title: Subchronic 90-day oral toxicity study in rats

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Amber liquid; Run No. 544; Drum No. 6131

METHOD

Method/guideline followed: This study was conducted according to OPPTS guidelines for the

conduct of a subchronic 90-d study (Organization for Economic Co-operation and Development 

(OECD) Guidelines for Testing Chemicals, ISBN 92-64-12221-4, adopted by the council at its 

535th meeting on 12 May, 1981).

Test type: Subchronic 90-Day Oral Toxicity Repeated-Dose

GLP compliant?: Yes

Year study performed: 1986

Test species: Sprague-Dawley rats from Charles River Laboratories, Wilmington, 

Massachusetts; Rats used in this study weighed 183-186 g (males) and 129-132 g (females) and 

were 41 days old at initiation.

Number of animals/sex/dose: 20

Route of administration: Oral - diet

Vehicle: None 

Exposure duration: 90 days (7 days/week)

Doses/concentrations tested: 0, 50, 125 or 320 ppm (0, 5, 12.5 and 32 mg/kg/day, respectively) 
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Statistical methods used: Evaluation of equality of means was made by the one way analysis of 

variance using the F distribution to assess significance. If significant differences among the 

means were indicated, Dunnett's test was used to determine significant differences from control 

means. Analysis of discrete data where appropriate was conducted using non-parametric 

procedures. All raw data were summarized and statistically evaluated using LABCAT modules 

designed by Innovative Programming Associates, Inc., One Airport Place, Princeton, NJ 08540.

Test method/conditions remarks: Four groups of rats (20 males and 20 females per group) 

were initially fed with the test article at dose levels of 0, 50, 125 and 320 ppm, seven days per

week over a period of 90 days. Pharmacotoxic signs were recorded as they were observed, 

including the time of onset, degree and duration. Cageside observations included, but were not 

limited to changes in skin and fur, eyes and mucous membranes, and also respiratory, 

circulatory, autonomic and central nervous system, somatomotor activity and behavior patterns. 

Measurements were made of food consumption twice weekly, the diet was replenished with fresh

freezer stored diet at the time of food consumption and the rats were weighed weekly. At the end 

of the 90-day period all surviving animals were sacrificed by CO2 asphyxiation. Any weak or 

moribund animals were removed, sacrificed and necropsied during the study as deemed 

necessary by the study director and principle investigator.

Blood was drawn by percutaneous cardiocentesis. All animals were fasted overnight prior to the 

time of sacrifice. The following determinations were made on twenty rats (10/sex) prior to 

initiation. In addition, after 90 days of feeding, five (5) animals per sex per group were chosen by 

random number tables and the following hematological and clinical chemistry parameters were 

determined: hemoglobin, hematocrit, erythrocyte count, total and differential leukocyte counts

platelet count, calcium, phosphorus, chloride, sodium, potassium, fasting glucose, serum alanine 

aminotransferase, serum aspartate aminotransferase, gamma glutamyl transpeptidase, urea 

nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements.

Ophthalmological examinations using an ophthalmoscope were made prior to the administration 

of the test substance on all animals. At termination of the study 5/sex/group chosen from a table 

of random numbers were evaluated. If changes in the eyes were detected all the animals would 

be examined.

All animals were subjected to necropsy which included examination of the external surface of 

the body, all orifices, and the cranial, thoracic, abdominal and pelvic cavities and their contents. 

The following organs were weighed: liver, gonads, adrenal glands, brain and kidneys.

The following organs and tissues from the animals at the 90-day sacrifice were preserved in 10% 

neutral buffered formalin for future histopathological examination: all gross lesions, brainincluding sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/

parathyroid, thymus, lungs, trachea, heart, sternum with bone marrow, salivary glands, liver, 

spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymides, 

prostate, and, if present, seminal vesicles), aorta, skin, esophagus, nasal turbinates, stomach, 

duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative lymph node, 

mammary gland, thigh musculature, peripheral nerve, eyes, femur-including articular surface, 

spinal cord at three levels - cervical, midthoracic and lumbar and exorbital lachrymal glands.
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A full histopathologic evaluation of the above organs and tissues was evaluated in all control and 

high dose animals sacrificed at 90 days of treatment. All animals which died or were sacrificed 

in a moribund condition prior to completion of the study were also subjected to a full 

histopathologic evaluation of the above named tissues and organs. All gross lesions in

All animals were examined histologically.

RESULTS

Endpoint value(s): LOAEL = 50 ppm (5 mg/kg-bw/day; lowest dose tested) based on mottled 

kidneys, dilated pelvis, subcapsular cystic or presence of calculi, lobular or small livers, 

congested lungs, crystalline material in the gastrointestinal tract with gas distention, increased 

hematocrit levels in females, minimal to moderate multifocal degeneration of the acinar cells of 

the pancreas and increased splenic pigmentation in females; NOAEL = Not established.

Results remarks: Non-specific clinical signs were detected in a few rats from the control, low 

and mid dose groups. These included lesions and/or scab formations with alopecia. In addition,

piloerection, chromodacryorrhea and poor grooming were observed. The majority of the high 

dose rats of both sexes displayed nonspecific clinical signs of toxicity: abnormal stance, 

abnormal gait, flaccid body tone, ptosis, decreased activity, chromodacryorrhea, elevated gait, 

loss of skin elasticity, poor grooming, dyspnea, tremors, ataxia and morbidity. In general, these 

signs resulted in either moribund sacrifice or death.

Mean body weight of male and female rats in the high dose group (320 ppm) was statically lower 

than their respective controls on study Days 7 through 90 and 14 through 90, respectively. Body 

weights were decreased in the mid-dose males (125 ppm) on Days 77 through Day 90; females 

on Days 28 and 35, and from Day 49 through Day 90. There was no statistically significant 

difference in the group mean weights in low dose males or females compared to their respective 

controls. The high dose male and female body weight gains were significantly reduced on Day 7 

through Day 35. A statistically significant weight loss was recorded on Day 42 until sacrifice for 

both the high dose males and females. Mid dose male body weight gains were reduced on Days 

70 and 84. The mid dose females exhibited a statistically significant smaller body weight gain on 

Days 21, 28 and 84. In addition, the total gain for the high and mid dose groups were

significantly reduced.

Food consumption was statistically reduced from control males in the high dose groups on Days 

14, 21, 24, 28, 35 and 38. High dose females consumed less food on Day 17 through Day 52. 

Mid-dose male consumption was significantly reduced on Days 45, 59, 63, 73 and 90. Females 

from this dosage group were significantly reduced on Days 45, 52, 59, 63, 70, 73 and Day 80 

through 90. Control and low dose female food consumption were comparable. Low dose males, 

however, exhibited statistically significant increases in the amount of feed consumed on Day 3 

through Day 28 and on Day 35.

Twenty-seven rats in the high dose group died during the study. Thirteen were found dead and 

fourteen were moribund sacrificed. The majority of the necropsy observations were nonspecific, and included stomach lesions, dark discoloration of the liver, kidneys and/or genital 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

17

organs and intestines, emaciation and apparent dehydration, lung congestion, liver foci and lack 

of thymic tissue.

A pre-termination ocular examination was performed on all the remaining animals. Twenty 

ocular lesions were noted: 2, 1, 4 and 13 from control to high dose, respectively. With the 

exception of a focal cataract observed in one mid dose male, observations recorded on the 

control, low and mid-dose animals, were considered inconsequential of treatment. Observations 

noted in the high dose group included two rats displaying pale fundus, one with conjunctivitis, 

one multifocal cataract, one multifocal anterior cortical and equatorial cataract each from both 

eyes and three focal cataracts from the right eyes. These noted abnormalities may be considered 

to be a direct or indirect effect from test article administration.

Terminal necropsy of the surviving rats revealed similar observations in each respective

treatment group: mottled kidneys, dilated pelvis, subcapsular cystic or presence of calculi; 

lobular or small livers; congested lungs; crystalline material in the gastrointestinal tract with or 

without gas distention was observed in each treatment group. A few high dose rats, however, 

exhibited small spleens and a larger number of focal or linear stomach lesions. In general, the 

high dose animals appeared thin and dehydrated.

Mean kidney, liver, testes and brain weights were statistically lower than the control means in 

high dose males. Mean high dose male and female relative-to-body adrenal, kidneys and brain 

weights were significantly increased compared to the control groups. In addition, the mid dose 

male and female liver, kidney and female brain weights to body weight were statistically larger 

than the control values. High dose males also displayed significant increases in kidney, liver and 

testes weight when compared to the percent body weight.

Evaluation of the blood chemistry parameters revealed statistically significant increases in 

SGPT, SGOT and blood urea nitrogen values in the male and female high dose groups. In high 

dose males, mean serum albumin, globulin, calcium, phosphorus, creatinine and total protein 

levels were significantly lower than those of the controls. High dose females exhibited 

significant increases in GGTP and decreases in total protein content.

High dosed males exhibited statistically significant increases in erythrocyte and hemoglobin 

counts. High dose male hemoglobin content was also significantly lower than control males. 

Hematocrit and erythrocyte values were statistically higher in the high dose females. Leukocyte 

counts, platelet counts and mean corpuscular volume were lower high in high dose males. Low 

and mid dose females exhibited significantly larger hematocrit levels.

Treatment-related microscopic changes were present in the eyes, pancreas, bone marrow, liver, 

spleen, kidneys, and mesenteric lymph nodes of almost all male and female rats in the high dose. 

In addition, atrophy was present in the salivary gland, thymus, stomach, and genital organs of 

both sexes in this group. Treatment-related changes were present in the pancreas and spleen 

from the rats in the 50 and 125 ppm dose groups. In the high dose rats, there was a high 

incidence of bilateral cataractous change in the eyes, diffuse atrophy of the acinar cells of the 

pancreas, vacuolation of the islet cells of the pancreas, bone marrow depletion, renal tubular 

vacuolation (hydropic change), atrophy of many organs, lymphoid depletion of the spleen, 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

18

thymus and mesenteric lymph node, and increased pigmentation of the liver and spleen. 

Minimal to moderate multifocal degeneration of the acinar cells of the pancreas and increased 

splenic pigmentation in the females were present in the tissues examined from the rats receiving 

50 and 125 ppm of DETDA in the diet.

Based upon the results of the Subchronic 90 Day Oral Toxicity Study in Rats with diethyl 

toluene diamine (DETDA) dose levels will be selected for a 24-month oncogenicity study in rats.

CONCLUSIONS

The LOAEL was 50 ppm (5 mg/kg-bw/day), the lowest dose tested; a NOAEL was not 

established. 

STUDY RELIABILITY

[2] Reliable with restrictions. Specific test method/guideline followed was not specified.

REFERENCES

Naismith, R., and Matthews, R. 1987. Subchronic 90 Day Oral Toxicity Study in Rats. Study 

No.: PH 470-ET-001-86. Diethyl toluene diamine (DETDA). Performing Laboratory: 

Pharmakon Research International, Inc. Waverly, PA. Sponsor: Ethyl Corporation, Baton 

Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

19

Study 2: 14- or 28-Day repeated-dose oral toxicity study with 28-day recovery period in rats 

(Bolus and Mathews, 1989)

Title: Repeated-dose oral toxicity/reversibility study in rats - 28 Day

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear amber liquid; Lot No. 2-87 and Run No. 544, Drum No. 6131 

METHOD

Method/guideline followed: This study was conducted in compliance with the Good 

Laboratory Practices Regulations as stated in the EPA Good Laboratory Practice Standards 

[Subpart I, Part 792, Chapter I of Title 40, Code of Federal Regulations], as well as the 

Organisation for Economic Co-operation and Development (OECD) Guidelines for Testing 

Chemicals, ISBN 92-64-12221-4, adopted by the council at its 535th meeting on 12 May, 1981.

Test type: Subchronic 28-Day Oral Repeated-Dose

GLP compliant?: Yes

Year study performed: 1987

Test species: Sprague-Dawley rats from Charles River Laboratories, Wilmington, 

Massachusetts; Rats used in this study were 48 days old and weighed 202-211 g (males) and 

147-154 g (females) at initiation.

Number of animals/sex/dose: 15

Route of administration: Oral, dietary

Vehicle: None

Exposure duration: 14 or 28 days

Doses/concentrations tested: 0 (control), 50, 125 or 320 ppm (0, 5, 12.5 and 32 mg/kg/day, 

respectively) 

Statistical methods used: Evaluation of equality of means was made by the one way analysis of 

variance using the F distribution to assess significance. If significant differences among the 

means were indicated, Dunnett's test was used to determine significant differences from control 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

20

means. Tukey's test was used to determine significant differences between the DETDA lots at 

the same dose levels.

All raw data were summarized and statistically evaluated using LABCAT modules designed by 

Innovative Programming Associates, Inc., One Airport Place, Princeton, NJ 08540 or SYSTAT, 

a PC based statistical package designed by SYSTAT, Inc,, Evanston, IL 60201.

Test method/conditions remarks: A repeated dose study was conducted to evaluate the 

progression and reversibility of toxicity observed in the 90-day study, and to determine if 

toxicity differed between 2 different DETDA lots. The test articles, diethyl toluene diamine 

(DETDA), Lot # 2-87 and Run 544, Drum 6131 (test article in previous 90 d study), were 

incorporated into standard certified commercial laboratory rat diet and fed ad libitum to seven 

groups of Sprague-Dawley rats (3 treatment groups per test article) seven days per week for a

period of 14 or 28 days. Each of the seven dose groups (n=15 rats/sex/group) were fed the diet 

mixture at dose levels of 0 (control) ppm, 50 (low) ppm, 125 (mid) ppm or 320 (high) ppm of 

either DETDA lot. At the end of the 14- and 28-day treatment intervals, five randomly selected 

rats per sex per treatment group were necropsied. The remaining rats received untreated control 

diet for 28 additional days and served as the recovery/reversibility group. Signs of toxicity were

recorded as they were observed, including the time onset, degree and duration. Cageside 

observations included, but were not limited to, changes in skin and fur, eyes and mucous 

membranes, and also respiratory, circulatory, autonomic and central nervous system,

somatomotor activity and behavior patterns. Mortality checks were made daily and recorded. 

Twice weekly measurements were made of food consumption, the diet was replenished with 

fresh freezer stored diet, and the animals were weighed.

Blood was drawn by percutaneous cardiocentesis. All animals were fasted overnight prior to the 

time of sacrifice. The following determinations were made on ten rats (5/sex) prior to initiation. 

In addition, the following hematology and clinical chemistry parameters were made on all 

animals sacrificed approximately 24 hours after 14, 28 and 56 days: hemoglobin, hematocrit

erythrocyte count, total and differential leukocyte counts, platelet count, calcium, phosphorus, 

chloride, sodium, potassium, fasting glucose, serum alanine aminotransferase, serum aspartate 

aminotransferase, serum amylase, serum lipase, gamma glutamyl transpeptidase, urea nitrogen, 

albumen, blood creatinine, total bilirubin, total serum protein measurements and serum insulin.

Ophthalmological examinations using an ophthalmoscope were made prior to the administration 

of the test substance on Days 14 and 28 of treatment and at the end of recovery (Day 56) on all 

animals.

All animals were subjected to necropsy, which included examination of the external surface of 

the body, all orifices, the cranial, thoracic, abdominal and pelvic cavities and their contents. The 

following organs were weighed: liver, gonads, adrenal glands, brain and kidneys.

The following organs and tissues from the animals sacrificed after 14 and 28 days of treatment 

and after the 28 day recovery period were preserved in a suitable medium for future 

histopathological examination: all gross lesions, brain (including sections of medulla/pons), 

cerebellar cortex and cerebral cortex, pituitary, thyroid/parathyroid, thymus, lungs, trachea, 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, 

gonads, uterus, accessory genital organs (epididymis, prostate, and, if present, seminal vesicles), 

aorta, skin, esophagus, nasal turbinates, stomach, duodenum, jejunum, ileum, cecum, colon, 

rectum, urinary bladder, representative lymph node (mesenteric), mammary gland, thigh 

musculature, peripheral nerve, eyes, femur-including articular surface, spinal cord at three levels 

(cervical, midthoracic and lumbar) and exorbital lachrymal glands.

A full histopathologic evaluation of the following organs and tissues were performed in all 

control and 320 ppm dosed animals sacrificed approximately 24 hours after 14 and 28 days of 

treatment and after the 28 day recovery period: all gross lesions, lungs, liver, adrenal glands, 

kidneys, gonads, salivary gland, eyes, pancreas, spleen, mesenteric lymph node, thymus, sternum 

with bone marrow and adipose tissue deposition whenever possible.

In addition, the sternum with bone marrow, eyes, kidney, liver, mesenteric lymph node, 

pancreas, salivary gland, spleen, thymus, gross lesions and adipose tissue deposition was 

evaluated in low and mid dose groups.

RESULTS

Endpoint value(s): LOAEL (Run 544, Drum 6131) = 125 ppm (12.5 mg/kg-bw/day) based on 

increased relative testes weights, compound-related degenerative changes in the acinar pancreas, 

degeneration of islet cells of the pancreas, cachexia and diabetes in males; NOAEL (Run 544, 

Drum 6131) = 50 ppm (5 mg/kg-bw/day); LOAEL (Lot 2-87) = 50 ppm (5 mg/kg-bw/day) based 

on decreased platelet counts in males at the lowest dose level tested; NOAEL (Lot 2-87) = Not 

established

Results remarks: No clinical signs of toxicity were observed in female rats in any treatment 

group during the study. Clinical signs of toxicity were observed in the majority of the high dose

DETDA-Run 544-treated males beginning on or about Day 35 and continuing until study

termination. These signs included decreased activity, abnormal stance, abnormal gait, poor 

grooming, decreased body tone, piloerection and excessive defecation and scab formation.

No rats died on test prior to scheduled sacrifice.

Mean male body weights in the high dose groups were statistically lower than the control mean 

during the majority of the study. During the recovery period (Days 28-56), the high dose male 

Run 544 group continued to lose weight, while animals treated with Lot 2-87 at the same dose 

gained weight. Female body weights were significantly reduced in both high dose groups 

compared to the control mean at three intervals during the study.

Mean male body weight in 320 ppm Run 544 group were significantly lower than that of the 320 

ppm Lot 2-87 group mean during Days 49 through 56. No significant differences between the 

two lots were noted for the females.

The daily body weight gains were significantly reduced at several intervals for the males in both 

the Run 544 and Lot 2-87 treatment groups at each level when compared to the controls. Female 
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22

body weight gains were less severely affected however, significance was indicated when 

compared to the controls.

Statistically significant differences were also noted when the daily gains of the different DETDA 

treatment groups were compared. Trend-related significant differences were observed for the 

high dose males. A significantly lower value was noted for the male high dose Run 546 group on

Day 56, when compared to the Lot 2-87 treated rats. The total mean daily gain was also 

significantly reduced in the high dose Run 544 group. There were no such trend-related 

differences observed for the females.

The daily food consumption for the males was significantly reduced in both high dose DETDA 

groups at several time periods, when compared to the controls. There were no significant 

differences noted in food consumption of the males or females when groups treated with the 

different DETDA Lots were compared to each other by dose level.

Ocular examinations were performed on all animals prior to the scheduled sacrifices. Lesions 

noted at 14 and 28 days of DETDA treatment were considered inconsequential to the 

administration of either test article. The pre-termination ocular examination, performed on Day 

56, detected multifocal anterior cataracts in 2 high dose Run 544 male rats. These lesions 

suggest a possible ocular toxicologic effect of the test compound.

The majority of necropsy observations recorded at either the 14- or 28-day interim sacrifices

were non-specific in nature. These observations included enlarged mandibular lymph nodes, 

scabs on the skin, lung and liver abnormalities, enlarged mesenteric nodes, kidneys exhibiting 

cortical cyst or dilated pelvic areas, parovarian uterine cysts, slight uterine hydrometra and a red 

area on the tip of the heart. Observations recorded at the terminal sacrifice (Day 56) included 

similar observations of the lungs, livers and kidneys. In addition, changes which were specific to 

the high dose Run 544 male group included reduced adipose tissue, rough hair coat, small 

salivary, glands, small spleens, poorly visible, dark and small pancreata, small testes, seminal 

vesicles, epididymides, prostrate and gastrointestinal tract lesions. There were no similar 

DETDA related changes noted at necropsy for the females.

There were no statistically significant differences observed in the absolute male or female organ 

weights at the Day 14 or Day 28 necropsy. At the Day 56 terminal necropsy, the high dose Run 

544 male body weights, liver and testes weights were significantly reduced, when compared to 

the control group. No significant differences were noted for the females at Day 56.

Comparisons by dosage group between the DETDA materials indicated the high dose male body 

weights, absolute liver and testes weights in the Run 544 group were significantly lower than the 

Lot 2-87 group at the terminal (Day 56) necropsy.

A statistically significant increase in the high dose male 2-87 relative brain and testes to body 

weights was observed at the Day 14 necropsy, when compared to the controls. The mid dose 

Run 544 male relative testes weights were also significantly larger. There were no significant 

organ weight changes noted for the females, when the dosage groups were compared to the 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

23

controls at the Day 14 interim sacrifice. The male relative liver weights were significantly larger 

in both high dose DETDA treated groups at the Day 28 necropsy.

Significant increases were noted in both high dose DETDA treated female relative kidney

weights. In addition, the female high dose Run 544 brain and liver weights were significantly 

larger, when compared to the control group.

There were no significant differences observed for either the male or female relative organ to 

body weights, when comparisons were made between the different DETDA materials by dose 

level at the Day 14 or Day 28 interim sacrifice.

At terminal necropsy (Day 56), the high dose Run 544 males displayed significantly larger 

relative brain, kidney and adrenal to body weights as compared to the controls. Females in the 

high dose 2-87 group exhibited significantly larger relative kidney weights, when compared to 

the controls.

When comparisons between DETDA materials were analyzed, significantly lower relative brain 

and kidney to body weights were recorded in the Run 544-high dose treated males versus the 2-

87 males. No significant differences were noted for the females.

No statistically significant differences were observed for either the male or female relative organ 

to brain weights at the Day 14 or Day 28 interim sacrifices when compared to the control group. 

At the Day 56 necropsy, the percent liver to brain weight was significantly reduced in the high 

dose Run 544 treated males.

There were no significant differences noted for the relative organ to brain weights, when the 

different DETDA materials were compared by treatment level to each other at any of the 

scheduled sacrifices.

Significant differences between controls and DETDA treatment groups of the same dose at each 

scheduled sacrifice were detected for certain serum chemistries. The majority of the differences 

were noted in the Run 544 mid and high dose male and female dosage groups at the Day 28 

interim sacrifice and the Day 56 terminal sacrifice. There were however, significant differences 

between controls and low dose DETDA groups at each sacrifice.

Low dose Lot 2-87 male platelet counts were significantly lower than the controls at the Day 14 

interim necropsy. At Day 28, significant hematological differences from controls were high dose 

male and mid dose female platelet counts.

Comparisons of DETDA materials at the Day 28 interim sacrifices indicated the platelet counts 

for each Run, 544 DETDA treated female were significantly higher than the Lot 2-87 females.

At the terminal necropsy (Day 56), the high dose Run 544 males exhibited a significant increase 

in hemoglobin and decrease in leucocyte and platelet counts, comparing controls alone.
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24

When comparisons were made between DETDA formulations, the hemoglobin values were 

significantly higher and the leucocyte counts lower in the high dose Run 544 males.

Compound-related degenerative changes in the acinar pancreas at 125 ppm and 320 ppm in 

males and 320 ppm in females were detected in rats exposed to DETDA 2-87 and DETDA Run 

544 Drum 6131 for twenty-eight days. A similar pancreatic acinar degeneration was not present 

at the 50 ppm dosage for either compound. Male rats were more severely affected and at an 

earlier time than the female rats. The 28-day recovery period allowed for an almost complete 

recovery from the pancreatic effects of DETDA 2-87. However, the pancreatic changes in the 

males exposed to DETDA Run 544 Drum 6131 progressed to degeneration of the islet cells of 

the pancreas and secondary changes of cachexia and diabetes.

In summary, the results of this study should be evaluated with consideration for those of the 

previous 90-d study. The results of the two studies indicate the target organ to be the pancreas. 

Male rats were more severely affected and at an earlier time and lower dose than females. 

Histologically, pancreatic acinar cells were affected first; islet cell involvement followed 

afterward in a time and dose dependent manner. Pathology in other organs occurred after or in 

conjunction with islet cell involvement and appeared secondary to the metabolic changes 

induced by the pancreatic toxicity. Secondary effects included ocular cataracts in animals with 

histologic evidence of islet cell toxicity. It was postulated these cataracts were associated with 

and related to hyperglycemia and thus the pancreatic toxicity. Toxicity of DETDA in the rat 

may vary with lot. A difference in toxicity between lots was observed. After exposure for 28 

consecutive days, DETDA Lot 2-87 exerted an effect, which was less toxic, than the Run 544, 

Drum 6131 preparation.

CONCLUSIONS

Based primarily on the severity and progressive treatment-related histopathological changes of 

the rats with the Run 544, Drum 6131 material and secondly the correlated alterations on 

chemical chemistry, it appears that the DETDA Lot 2-87 formulation was less toxic than the Run 

544, Drum 6131 preparation.

The LOAEL and NOAEL for Run 544, Drum 6131 were 125 and 50 ppm (12.5 and 5 mg/kgbw/day), respectively. The LOAEL for Lot 2-87 was 50 ppm (5 mg/kg-bw/day), the lowest dose 

tested; a NOAEL for Lot 2-87 was not established. 

STUDY RELIABILITY

[2] Reliable with restrictions. Details were missing from the study summary including purity 

and composition of the test substance and test method/guideline followed.

REFERENCES

Bolus, G., and Matthews, D. 1989. Repeated Dose Oral Toxicity/Reversibility Study in Rats -

28 Day. PH 436-ET-001-87. Diethyl toluene diamine (DETDA). Performing Laboratory: 

Pharmakon Research International, Inc. Waverly, PA. Sponsor: Ethyl Corporation, Baton 

Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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Study 3: 28-Day repeated-dose oral toxicity study in rats (Naismith, 1986)

Title: Repeated dose oral toxicity study in rats – 28 day dose-range-finding

This study is: Adequate Not Adequate

This study is not adequate because it is a range-finding study, in which clinical chemistry, 

hematology and histopathology analyses were not performed. Additionally, the test guideline 

used was not indicated.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Trade name: ETHACURE 100 Curative; Physical state: Amber-colored liquid; Lot: 

Run #544, Drum # 6131

METHOD

Method/guideline followed: Not indicated

Test type: Subchronic 28-Day Oral Repeated-Dose

GLP compliant?: Yes

Year study performed: 1986 

Test species: Sprague-Dawley rats

Number of animals/sex/dose: 5

Route of administration: Oral - diet

Vehicle: None

Exposure duration: 28 days (7 days/week)

Doses/concentrations tested: 0, 40, 400, 800, 1200, 1600 or 3200 ppm (0, 4, 40, 80, 120, 160 

and 320 mg/kg-bw/day, respectively)

Statistical methods used: Evaluation of equality of means was made by the one way analysis of 

variance using the F distribution to assess significance (p ≤ 0.05). If significant differences 

among the means were indicated, Dunnett’s test was used to determine significant differences 

from control means (p ≤ 0.05).
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

26

Test method/conditions remarks: A 28-day dose range finding study was conducted in rats to 

select dose levels for a 90-day study. The commercial DETDA product (Run 544) was mixed 

with standard certified commercial laboratory rat diet and fed ad libitum to seven groups of 

Sprague-Dawley rats, seven days per week for 28 days (except where noted below). Each of 

seven groups (n=5 rats/sex/dose) was fed the diet mixture at dose levels of 0, 40, 400, 800, 1200, 

1600 or 3200 ppm DETDA. Animals were observed daily for clinical signs of toxicity. Body 

weights and food consumption were measured weekly. All animals were subjected to a gross 

necropsy. No clinical chemistry, hematology or histopathology was performed in this dose 

range-finding study. This study was conducted according to U.S. FDA, U.S. EPA, and OECD 

Good Laboratory Practices.

RESULTS

Endpoint value(s): The LOAEL was 400 ppm (40 mg/kg-bw/day) and the NOAEL was 40 ppm 

(4 mg/kg-bw/day) in both males and females.

Results remarks: Clinical signs of illness were not observed in the control group. No specific 

test article-related clinical signs were seen at the 40 or 400 ppm doses. Clinical signs of toxicity 

were observed in the higher doses, with the incidence and severity increasing in a dose 

dependent manner with study duration. One rat (female) died at the 3200 ppm dose level. 

Necropsy revealed a fluid-filled, reddish-brown, gastro-intestinal tract. Due to extreme weight 

loss and clinical signs, the remaining rats in the 3200 ppm dose group were sacrificed on day 17.

Mean female body weights were significantly lower than the control mean throughout the study 

in the 800, 1200 and 1600 ppm dose groups. Mean female body weight in the 400 ppm group 

was statistically reduced during the final week of the study. Mean body weight of the 40 ppm 

group was comparable to the control mean. Body weights of the male rats were similarly 

reduced during the study, although statistical significance was observed only on Day 3 and 

weeks 3 and 4 at the 800 ppm level. Mean body weights in the 3200 ppm dose group (both male 

and female) were statistically reduced from that of the controls until sacrifice.

Mean food consumption in males in the 800 to 3200 ppm groups was statistically and/or dose 

dependently reduced from that of the controls throughout the study. Statistically significant 

differences between treated and control animals were observed infrequently at the 400 ppm level. 

Male food consumption in the 0 and 40 ppm groups was statistically comparable. Food 

consumption of female rats was statistically reduced at the 800, 1200, 1600 and 3200 ppm dose 

groups in a similar manner as the males.

No noteworthy abnormalities were recorded at terminal necropsy in the control rats with the 

exception of a dislocated upper incisor and chromodacryorrhea. Infrequent mottled kidneys, pale 

kidneys, skin lesions with scab formation, congested lungs, white foci or clear foci on the lungs 

were recorded in a few rats in the treatment groups. In general, there were no test article-related 

lesions recorded at terminal necropsy in any of the treated animals.

At terminal necropsy the kidneys and livers were weighed. Male relative liver-to-body weights 

were statistically increased at 1200 and 1600 ppm. There was a statistically significant increase 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

27

in the mean relative kidney weights at the 1200 ppm level. No statistically significant 

differences for relative organ weights were observed in the female rats.

CONCLUSIONS

The LOAEL in a 28-day repeated-dose oral study in Sprague-Dawley rats was 400 ppm (40 

mg/kg-bw/day) in males and females, based on reduced food consumption in both sexes and 

reduced body weight in females. The NOAEL was 40 ppm (4 mg/kg-bw/day) in males and 

females.

STUDY RELIABILITY

[3] Not reliable. In this range-finding study, clinical chemistry, hematology and histopathology 

analyses were not performed. Additionally, the test guideline used was not indicated. 

REFERENCES

Naismith, R. 1986. Repeated dose oral toxicity study in rats – 28 day dose-range-finding. PH 

436-ET-001-86. Diethyl toluene diamine (DETDA). Performing Laboratory: Pharmakon 

Research International, Inc. Waverly, PA. Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

28

Study 4: Repeated-dose dermal toxicity study in rabbits (Lamb et al., 1981)

Title: Subchronic 21-day dermal toxicity study on diethyltoluenediamine (DETDA) in rabbits

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 95.78% Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Referred to as “diethyltoluamine” (DETA)

Remarks: Brown liquid; Isomer Ratio: 80:20 2,4-DETDA:2,6-DETDA 

METHOD

Method/guideline followed: Not indicated

Test type: Subchronic dermal toxicity

GLP compliant?: Not indicated

Year study performed: 1980

Test species: New Zealand White rabbits. 2.31-3.59 kg (males) and 2.40-3.17 kg (females) at 

study start.

Number of animals/sex/dose: 10

Route of administration: Dermal

Vehicle: None. Test substance was applied undiluted.

Exposure duration: 6 hours daily, 5 d/wk for three weeks

Doses/concentrations tested: 0 (untreated control), 1, 10, 100 mg/kg-bw/day

Statistical methods used: The Waller-Duncan k-ratio t-test was used to compare results of 

treated rabbits to those of concurrent controls. Differences between the results in a particular 

group before and after treatment were tested using Student’s t-test.

Test method/conditions remarks: Young adult male and female New Zealand White rabbits 

were used to examine the repeated dose toxicity of DETDA when administered dermally. 

Groups of 20 rabbits (10/sex/dose) were treated dermally with 0, 1, 10 or 100 mg/kg-bw once 

daily, five days per week, for a total of 15 doses. 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

29

The backs of the rabbits were shaved free of hair using electric clippers with an angra head. The 

last five rabbits of each sex on each level were abraded on the shaved area using a nylon bristle 

brush. Rabbits were shaved and abraded weekly, at least the day before application of the next 

dose or earlier. At the time of dosing, plastic collars were placed on the animals. The volume of 

material to be applied was determined from the animals’ most recent body weight and was 

applied undiluted to the shaved area on the back. Dosage volumes were measured and applied 

using a 5 μl Hamilton syringe for level I, a 50 μl Hamilton syringe for level II and a 500 μl 

Hamilton syringe for level III. The material was then covered with a square of gauze large 

enough to cover the liquid, two single layers thick, which was secured with surgical 

hypoallergenic adhesive tape. The gauze was covered with a larger square of clear plastic 

secured with adhesive tape. The animals were returned to their cages after application of the test 

material. After six hours of exposure, the plastic, gauze and tape were removed and the treated 

area was wiped with gauze to remove any remaining test material. The collar was removed and 

the animal was returned to the cage.

The animals were observed twice daily on weekdays when material was applied and removed for 

the appearance of toxicological and pharmacological signs; animals were observed once daily on 

weekends. Dermal irritation readings according to the method of Draize were made daily 

immediately prior to dosing, including the first day of treatment. Body weights were determined 

at the beginning of the test and every three to four days thereafter. Surveillance was made to 

preclude loss of animals due to autolysis, misplacement or other management problems.

Animals were assigned randomly to test or control groups. The first five animal numbers of each 

sex in each test and control group were then selected for clinical laboratory testing both before 

initiating the study and on the day of the last treatment.

Hematology tests included determination of hematocrit and hemoglobin concentration, red and 

white blood cell counts, differential white cell counts and platelet counts.

Blood chemistry tests included determinations of calcium, sodium, potassium, glucose, blood 

urea nitrogen, total bilirubin, total cholesterol, albumin, globulin and total protein concentrations 

and of serum lactic dehydrogenase, serum glutamic pyruvic transaminase, serum glutamic 

oxaloacetic transaminase and serum alkaline phosphatase activities.

All test and control animals were subjected to gross necropsy. The liver, kidneys, heart, gonads, 

thyroid (with parathyroid), adrenals, lungs, brain and pituitary were weighed. Tissues were 

collected and fixed in 10% buffered formalin for histopathology examination.

RESULTS

On the first day of treatment, it was observed that a male rabbit to be treated with 100 mg/kg-bw 

(no. 434) had not eaten for several days. On that same day, a possible replacement, No. 490, was 

designated. No. 490 was shaved and treated; a blood sample was collected prior to treatment. 

On day 3 of treatment, No. 434 appeared thin and had not resumed eating. The results of the 

clinical studies on the blood sample from No. 490 were normal, so No. 490 replaced No. 434 on 

day 3 of treatment. Since the cessation of food consumption by No. 434 occurred prior to 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

30

treatment, its condition could not have been related to treatment. No mortality occurred in 

treated rabbits.

No significant differences were apparent among mean body weights of either sex throughout the 

study. However, two female rabbits receiving 100 mg/kg-bw and one receiving 10 mg/kg-bw 

failed to gain weight throughout the study. All of these showed initial weight gain followed by 

loss. Of the two rabbits receiving 100 mg/kg-bw, one failed to regain her pretest body weight, 

while the other regained to 0.02 kg higher than her pretest body weight. The female receiving 10 

mg/kg-bw exhibited only approximately half of the total average weight gain of her group. No 

differences in rate of weight gain were apparent among male rabbits treated at this dosage. 

No behavioral abnormalities were observed in any of the rabbits during the study. Occasional 

coughing, sneezing and/or ocular discharge were observed in some of the rabbits, including 

controls, and were considered incidental.

No evidence of skin irritation was observed in control rabbits of either sex. Slight erythema 

accompanied occasionally by slight edema was observed sporadically in some rabbits at 1 

mg/kg-bw. Rabbits at 10 mg/kg-bw exhibited erythema and some edema, and the incidence of 

these was consistent by the third week of treatment. One female rabbit at 10 mg/kg-bw exhibited 

a scab in the treated area of the skin in the third week of treatment. Rabbits at 100 mg/kg-bw 

exhibited erythema accompanied by very slight to moderate edema through most of the treatment 

period. The severity of the edema increased during the study. Open wounds were observed in 

the skin of two males and two females of this group and small scabs were observed on one male 

and two females with no prior evidence of skin injury.

Occasional small differences in means of hematology results were observed among groups 

before treatment; however, the results for all the groups were within the range of normal 

biological variation. In the results after treatment occasional differences in hematology values 

were observed among groups of male rabbits, but these were not outside the range of normal 

biological variations. In female rabbits, a dose-related decrease in monocyte counts was 

observed; the counts were significantly different from control females for those receiving 10 and 

100 mg/kg-bw DETDA but not for those receiving 1.0 mg/kg-bw. However, the counts in these 

two groups were within the range of counts (0-11) observed in rabbits of the same sex and 

appropriate age. No other differences of biologic significance were observed in female rabbits.

In rabbits before treatment no differences in serum chemistry results between groups were 

observed that were outside the range of normal biological variation. In rabbits after treatment, 

dose-related decreases in serum calcium and sodium concentrations were observed in both male 

and female rabbits. The calcium concentrations were statistically significantly different from 

control in females receiving 10 or 100 mg/kg-bw DETDA and in males receiving 100 mg/kg-bw. 

The sodium concentrations were statistically significantly different from control in all treated 

groups of females and in males receiving 10 mg/kg-bw DETDA but not those receiving 100 

mg/kg-bw. Decreased serum potassium concentrations were observed in male, but not female, 

rabbits; however, the results were not significantly different from control. The cause of the 

decreases in serum electrolytes was not apparent. Although the decreases were statistically 

significant and dose-related, none of the electrolytes decreased to levels likely to impair normal 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

31

physiologic processes. No other differences in chemistry results in male or female rabbits were 

observed that were outside the range of normal biological variation.

Chronic dermatitis was observed in treated skin of control and treated rabbits of either sex. The 

incidence and severity were greater in treated rabbits than in controls, however. The incidence 

of dermatitis increased with increasing dosage of DETDA. The severity of the lesions increased 

with dosage in female rabbits but was essentially constant in treated male rabbits. Because 

dermatitis was observed only occasionally in untreated skin, it was concluded that the dermatitis 

on treated skin was a direct result of applications of DETDA. 

Cysts were observed in thyroid glands of rabbits of either sex. The incidence was dose-related in 

males but not in females. These lesions are common histopathologic findings in rabbits of this 

age and strain, however, and the cysts observed were probably not a consequence of treatment. 

The remaining lesions observed occurred randomly in treated and control groups and were 

considered incidental.

Endpoint value(s): LOAEL (systemic toxicity) > 100 mg/kg-bw-bw/day based on no significant 

effects at the highest dose tested; NOAEL = 100 mg/kg-bw-bw/day. LOAEL (application site 

irritation) = 10 mg/kg-bw-bw/day based on mild to moderate irritation; NOAEL = 1 mg/kgbw/day

Results remarks: None specified

CONCLUSIONS

Repeated dermal application of diethyltoluamine at 1.0 mg/kg-bw resulted in very slight 

irritation of no biologic significance. Application of 10 and 100 mg/kg-bw resulted in mild to 

moderate local irritation and open wounds were observed in skin of some rabbits treated at 100 

mg/kg-bw. No other significant overt toxicity was observed in treated rabbits.

Decreases in serum calcium and sodium concentrations were observed in some treated groups of 

either sex; however, the electrolyte concentrations were not reduced sufficiently to interfere with 

normal physiologic processes.

No abnormalities that could be related to treatment were observed at gross necropsy. 

Histopathologic examination of fixed tissues revealed dermatitis in treated skin; no other lesions 

that could be related to treatment were observed.

Repeated application of DETDA at 1.0, 10 or 100 mg/kg-bw resulted in no significant systemic 

effects. Application of 10 and 100 mg/kg-bw resulted in significant local effects on treated skin; 

these effects were more pronounced at the highest dosage. No significant local effects were 

observed after application of DETDA at 1.0 mg/kg-bw.

STUDY RELIABILITY
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

32

[2] Reliable with restrictions. The 21-day duration of this study is shorter than the 28-day 

duration needed to adequately address the repeated-dose endpoint. Additionally, test 

method/guideline followed and GLP compliance was not specified. 

REFERENCE

Lamb, D., Hixson, E., Brewer, G., Hoss, H., Mallicoat, D., and Schroeder, R. 1981. Subchronic 

21-Day Dermal Toxicity Study on Diethyltoluamine (Diethyltoluenediamine, DETA) in Rabbits. 

Study Number 80-123-02. Performing Laboratory: Stanley Research Center, Stilwell, Kansas. 

Sponsor: Mobay Chemical Corporation.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

33

C-6. Genetic Toxicity – Gene Mutation: Test Requirement (E1) under Final HPV2 Test 

Rule Dated January 7, 2011

Summary of endpoint: Adequate 

Study 1: Mutagenicity studies in Salmonella typhimurium (Barfknecht and Naismith, 1986)

Title: Ames Salmonella/microsome plate test

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Referred to as DETDA-OBG, Lot #5868-1; Amber liquid; Composition: 0.7% 

METDA, 78.2% 2,4-DETDA, 20.3% 2,6-DETDA, 0.8% other isomers, trialkylated 

toluenediamine (as cited in Mulholland, 1986).

METHOD

Method/guideline followed: Protocol- 301 84 EPA/OECD

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1986

Test species: Salmonella typhimurium strains TA1535, TA1533, TA1538, TA98 and TA100

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 100, 330, 1000, 3300 and 10,000 μg/plate (preliminary toxicity screen); 

0 (DMSO solvent control), 60, 200, 600, 2000 and 6000 μg/plate (plate incorporation assay)

Statistical methods used: In most tests with the Salmonella/Microsome Assay, results are 

either clearly positive or clearly negative. A positive result is defined as a reproducible, doserelated increase in the number of histidine-independent colonies with at least one dose point 

inducing a mutant frequency value that is two-fold the solvent control value. Significance at the 

95% confidence limit is determined by the program developed by Moore and Felton. This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

34

the 95% confidence limits of the historical mean for control values and the test chemical 

produces the highest increase equal to or greater than three times the solvent control value, the 

test chemical is considered positive, A negative result is defined as the absence of a reproducible 

increase in the number of histidine-independent colonies.

Test method/conditions remarks: 

Negative and Positive Controls: Tester strains TA1535, TA1537, TA1538, TA98 and TA100 

were plated in triplicate with the appropriate solvent (DMSO), both with and without metabolic 

activation, to obtain background lawn and revertant colony formation to serve as negative 

solvent controls. In order to validate the integrity of the test system, all tester strains were also 

run, in triplicate, with known positive response chemicals. Positive controls not requiring 

metabolic activation were strain specific and were as follows: sodium azide (10 μg/plate) for 

TA1535 and TA100; 9-aminoacridine (150 μg/plate) for TA1537; 2-nitrofluorene (5.0 μg/plate) 

for TA1538 and TA98

The positive control requiring metabolic activation was 2-anthramine (2-aminoanthracene) (2.5 

μg/plate) in all strains. 

Preliminary Toxicity Screen: The preliminary toxicity screen for the Ames Assay performed 

without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, 

TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels 

the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test 

compound was prepared to a concentration of 100 mg/ml in DMSO at the request of the sponsor 

and five different levels tested for toxicity (100, 330, 1000, 3300 and 10,000 μg/plate). Top agar, 

used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine 

- 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass 

tubes with kaputs were labeled and placed into a Fisher IsotempR Dry Bath at 45°C. All control 

and treated tubes and plates were done in duplicate. Using sterile technique, the following were 

added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the 

appropriate concentration of the test compound. The tubes were vortexed and poured onto 

minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay 

was allowed to harden. The same procedure was repeated for each tester strain, within an hour 

the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 

hours following which the background lawn and spontaneous revertants were observed and 

scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence 

of pindot colonies and the absence of a confluent lawn of bacteria.

Plate Incorporation Assay: The test article, DETDA-OBG, Lot #5868-1, was evaluated in the 

five Ames/Salmonella tester strains TA1535, TA1537, TA1538, TA98 and TA100 at the dose 

levels of 60, 200, 600, 2000 and 6000 μg/plate with and without the addition of a rat liver 

derived metabolic activation preparation. There was 0.10 ml of S-9 supernatant (44 mg 

protein/ml) per 1.0 ml of the S-9 mix in the rat liver metabolic activation preparation.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

35

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher IsotempR Dry 

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All 

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of 

tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation had, in addition to the 

preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats. 

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 

strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.

Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. A summary of the data is 

presented in the summary data sheet contained in this report.

RESULTS

Preliminary Toxicity Screen: There were no revertant colonies observed for strain TA100 at the 

high dose level of 10,000 μg/plate. There were few revertant colonies observed at the high 

concentration in strain TA1538. Normal bacterial lawn growth and revertant colony numbers 

were observed at the other dose levels for both tester strains. Therefore, a dose of 6000 μg/plate 

was chosen as the high concentration for the Plate Incorporation Mutation Assay.

Plate Incorporation Assay: The test article did not induce increases of mutant frequency in the 

five Ames/Salmonella tester strains TA1535, TA1537, TA1538, TA98 and TA100 without 

metabolic activation preparation at the dose levels of 60, 200, 600, 2000 and 6000 μg/plate.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

36

The test article induced a statistically significant (at the 95% confidence limit) linear mutational 

dose response in the five Ames/Salmonella tester strains TA1535, TA1537, TA1538, TA98 and 

TA100 with metabolic activation preparation at dose levels of 60, 200, 600, 2000 and 6000 

μg/plate. The test article was retested in the five tester strains at dose levels of 500, 1500, 3000, 

6000 and 8000 μg/plate with metabolic activation preparation and the positive findings were 

confirmed.

In strain TA1535 of Salmonella typhimurium, a value that was 2.2-fold the mean solvent control 

mutant frequency value was observed at a dose level of 6000 μg/plate in the initial assay. A 

mean mutant frequency value that was 2.9-fold the solvent control value was observed for tester 

strain TA1537 at a concentration of 6000 μg/plate in the confirmatory assay. A value that was 

28.3-fold the background mutant frequency occurred at a dose level of 6000 μg/plate for strain 

TA1538 in the second mutation assay. In the initial assay, a value that was 8.9-fold the solvent 

control value was observed at the 6000 μg/plate level for strain TA98. In strain TA100, a 

maximum fold increase of 3.7-fold over the solvent control mutant frequency value was 

observed at the 6600 μg/plate dose level in the second mutation assay. 

Genotoxicity result: Positive in the presence of metabolic activation

Cytotoxic concentration (if applicable): 6000 μg/plate

Results remarks: The solvent and positive controls employed in the evaluation of the test 

article induced mutant frequency values that were within the acceptable limits of mean historical 

data.

CONCLUSIONS

The test article, DETDA-OBG, Lot #5868-1, induced a positive mutational response in all five 

Ames/Salmonella tester strains in the presence of a rat liver derived metabolic activation 

preparation. The results suggest that it is a metabolite(s) of the test article that is responsible for 

the mutagenic response to the five tester strains.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

Barfknecht, T., and Naismith, R. 1986. Ames Salmonella/Microsome Plate Test with DETDAOBG Lot #58681 (EPA/OECD). Report # PH 301-ET-002-86. Sponsor: Ethyl Corporation, 

Baton Rouge, LA. 

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

37

Study 2: Mutagenicity studies in Salmonella typhimurium (Barfhecht et al., 1986)

Title: Ames Salmonella/microsome plate test (EPA/OECD)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 98.9% 3,5-Diethyltoluene-2,6-diamine, CAS No. 2095-01-4; Synonym: 2,6-DETDA

Remarks: Referred to as 2,6 DETDA Isomer #2, Lot #5712-71-6; Pale tan solid; Composition: 

98.9% 3,5-diethyltoluene-2,6-diamine, 0.8% monoethyltoluenediamine, 0.3% other isomers (as 

cited in Mulholland, 1986)

METHOD

Method/guideline followed: Protocol-301 84 EPA/OECD

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1986

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 100, 330, 1000, 3300 and 10,000 μg/plate (preliminary toxicity screen); 

60, 200, 600, 2000 and 6000 μg/plate (mutation assay)

Statistical methods used: In most tests with the Salmonella/microsome Assay, results are either 

clearly positive or clearly negative, A positive result is defined as a reproducible, dose-related 

increase in the number of histidine-independent colonies with at least one dose point inducing a 

mutant frequency valve that is two-fold the solvent control value. Significance at the 95% 

confidence Limit is determined by the program developed by Moore and Felton (1983). This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 

the 95% confidence limits of the historical mean (See Historical Data) for control values and the 

test chemical produces the highest increase equal to or greater than three times the solvent 

control value, the test chemical is considered positive. A negative result is defined as the absence 

of a reproducible increase in the number of histidine-independent colonies.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

38

Test method/conditions remarks:

Negative and Positive Controls: Tester strains TA1535, TA1537, TA1538, TA98 and TA100 

were plated in triplicate with the appropriate solvent, both with and without metabolic activation, 

to obtain background lawn and revertant colony formation to serve as negative solvent controls. 

In order to validate the integrity of the test system, all tester strains were also run, in triplicate, 

with known positive response chemicals. Positive controls not requiring metabolic activation 

were strain specific and were as follows:

TA1535 - without activation - Sodium azide (10 μg/plate)

TA1537 - without activation - 9-aminoacridine (150 μg/plate)

TA1538 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA98 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA100 - without activation - Sodium azide (10 μg/plate)

The positive control requiring metabolic activation was 2-anthramine (2-Aminoanthracene) (2.5 

μg/plate) in all strains.

Preliminary Toxicity Screen: The preliminary toxicity screen for the Ames Assay performed 

without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, 

TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels 

the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test 

compound was prepared to a concentration of 100 mg/ml and five different levels tested for 

toxicity (100, 330, 1000, 3300 and 10,000 μg/plate). Top agar, used as an overlay, was 

reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a 

volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs 

were labeled and placed into a Fisher IsotempR Dry Bath at 45°C. All control and treated tubes 

and plates were done in duplicate. Using sterile technique, the following were added to each 

tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate 

concentration of the test compound. The tubes were vortexed and poured onto minimal glucose 

plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to 

harden. The same procedure was repeated for each tester strain. Within an hour the plates were 

inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following 

which the background lawn and spontaneous revertants were observed and scored as normal 

growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies 

and the absence of a confluent lawn of bacteria.

Plate Incorporation Assay: Test article, 2,6 DETDA # 2, Lot #5712-71-6, was evaluated in 

strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and 

without metabolic activation preparation at dose Levels of 60, 200, 600, 2000 and 6000 μg/plate. 

There was 0.10 ml of S-9 supernatant (44 mg protein per ml) per 1.0 ml of S-9 mix in the rat 

liver metabolic activation preparation.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher lsotempR Dry 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

39

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquot of top agar solution, 0.1 ml of tester 

strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation had, in addition to the 

preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats.

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 

strain within an hour the plates were inverted and placed in a dark 37°C incubator. The plates 

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay, The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.

Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. 

RESULTS

There were no observed increases in mutation frequencies in strains TA1535, TA1537, TA1538, 

TA98 and TA100 with and without metabolic activation, at dose levels of 60, 200, 600, 2000 and 

6000 μg/plate. The high dose of 6000 μg/plate appeared to be toxic to the five tester strains as 

evidenced by a reduction in the mean number of revertant colonies relative to the solvent control 

values. 

Genotoxicity result: Negative 

Cytotoxic concentration (if applicable): ≥ 3300 μg/plate

Results remarks: All solvent and positive controls employed in the evaluation of the test article 

induced mean mutant frequency values, which were within the acceptable range of mean 

historical data.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

40

CONCLUSIONS

The test substance was not mutagenic in Salmonella typhimurium strains TA1535, TA1537, 

TA1538, TA98 or TA100 with or without metabolic activation.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCES

Barfhecht, T., Naismith, R., and Matthews, R. 1986. Ames Salmonella/Microsome Plate Test 

with 2,6 DETDA #2 Lot #5712-71-6 (EPA/OECD). Report # PH 301-ET-003-86. Performing 

Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

41

Study 3: Mutagenicity studies in Salmonella typhimurium (Barfknecht et al., 1985a)

Title: Ames Salmonella/microsome plate test (EPA/OECD)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Referred to as 5711-75-7 (Orangeburg sample); Red orange liquid; Composition: 

2.805% METDA, 76.082% 2,4-DETDA, 20.480% 2,6-DETDA, 0.260% 2,4,6-TETDA, 0.285% 

trialkylated PDA, 0.089% others (as cited in Mulholland, 1986).

METHOD

Method/guideline followed: Protocol-301 84 EPA/OECD

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1985

Test species: Salmonella typhimurium strains TA1538, TA98 and TA100

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 100, 333, 1000, 3333 and 10,000 μg/plate

Statistical methods used: In most tests with the Salmonella/microsome Assay, results are either

clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related 

increase in the number of histidine-independent colonies with at least one dose point inducing a 

mutant frequency valve that is two-fold the solvent control value. Significance at the 95% 

confidence limit is determined by the program developed by Moore and Felton (1983). This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 

the 95% confidence limits of the historical mean (See Historical Data) for control values and the 

test chemical produces the highest increase equal to or greater than three times the solvent 

control value, the test chemical is considered positive. A negative result is defined as the absence 

of a reproducible increase in the number of histidine-independent colonies.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

42

Test method/conditions remarks:

Negative and Positive Controls: Tester strains TA1538, TA98 and TA100 were plated in

triplicate with the appropriate solvent, both with and without metabolic activation, to obtain 

background lawn and revertant colony formation to serve as negative solvent controls. In order 

to validate the integrity of the test system, all tester strains were also run with known positive 

response chemicals, six (6) plates being seeded per point.

The positive control requiring metabolic activation was 2-anthramine (2-Aminoanthracene) (2.5 

μg/plate) in all strains.

Plate Incorporation Assay: Test article, 5711-75-7, was received as a red orange liquid and was 

dissolved in dimethylsulfoxide (DMSO) at the suggestion of the sponsor. The dose levels 

evaluated in the Ames/Salmonella mutation Assay with tester strains TA1538, TA98 and TA100 

in the presence of metabolic activation preparation were 100, 333, 1000, 3333 and 10,000 

μg/plate. There was 0.08 ml of S-9 fraction (35.7 mg protein per ml) per 1.0 ml of rat liver 

derived S-9 metabolic activation preparation. Plates were scored after approximately 48 hours 

and 72 hours of incubation at 37°C ± 2°C.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher IsotempR Dry 

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquot of top agar solution, 0.1 ml of tester 

strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: A S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats was employed for all test conditions.

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 

strain within an hour the plates were inverted and placed in a dark 37°C incubator. The plates 

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition. Three plates from each set of six were scored at 48 and 72 hours of incubation. The 

plates were also observed for unusual colony morphology.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay, The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

43

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.

Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. 

RESULTS

The test article induced statistically significant (at the 95% confidence interval) linear mutational 

dose responses in Ames/Salmonella tester strains TA1538, TA98 and TA100 in the presence of

metabolic activation preparation over the dose range of 100-10,000 μg/plate at approximately 38 

and 72 hours of incubation. The maximum observed fold-increase over the mean solvent control 

mutant frequency values which occurred after the 48 hour incubation period for strains TA1538 

and TA98 were 5.6 and 3.8, respectively. A mutant frequency value that reached 1.9-fold the 

mean solvent control value for strain TA100 occurred after the 72 hour incubation period. No 

unusual colony morphology was observed at either 48 or 72 hours of incubation. 

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): N/A

Results remarks: All solvent and positive controls employed in the evaluation of the test article 

induced mutant frequency values that were within the acceptable limits of mean historical data 

except the 72 hour incubation solvent control value for strain TA98. The sponsor requested that

the laboratory not repeat strain TA98.

CONCLUSIONS

The test substance was mutagenic in S. typhimurium strains TA1538 and TA98 in the presence 

but not the absence of metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study, including 

information on cytotoxicity of the test substance. 

REFERENCES

Barfknecht, T., Naismith, R., and Matthews, R. 1985a. Ames Salmonella/Microsome Plate Test: 

5711-75-7 (EPA/OECD). Report # PH 301-ET-010-85. Performing Laboratory: Pharmakon 

Laboratories, Waverly, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

44

Study 4: Mutagenicity studies in Salmonella typhimurium (Barfknecht et al., 1985b)

Title: Ames Salmonella/microsome plate test (EPA/OECD)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 99.953% 3,5-Diethyltoluene-2,4-diamine, CAS No. 2095-02-5; Synonym: 2,4-

DETDA

Remarks: Lot No. 5712-38-7; Clear yellow liquid; Composition: 99.953% 3,5-diethyltoluene2,4-diamine, 0.023% 2,4,6-triethyltoluenediamine, 0.024% others (as cited in Mulholland, 1986).

METHOD

Method/guideline followed: Protocol-301 84 EPA/OECD

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1985

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 625, 1250, 2500, 5000 and 7500 μg/plate

Statistical methods used: In most tests with the Salmonella/microsome Assay, results are either 

clearly positive or clearly negative, A positive result is defined as a reproducible, dose-related 

increase in the number of histidine-independent colonies with at least one dose point inducing a 

mutant frequency valve that is two-fold the solvent control value. Significance at the 95% 

confidence limit is determined by the program developed by Moore and Felton (1983). This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 

the 95% confidence limits of the historical mean (See Historical Data) for control values and the 

test chemical produces the highest increase equal to or greater than three times the solvent 

control value, the test chemical is considered positive. A negative result is defined as the absence 

of a reproducible increase in the number of histidine-independent colonies.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

45

Test method/conditions remarks:

Negative and Positive Controls: Tester strains TA1535, TA1537, TA1538, TA98 and TA100 

were plated in triplicate with the appropriate solvent, both with and without metabolic activation, 

to obtain background lawn and revertant colony formation to serve as negative solvent controls. 

In order to validate the integrity of the test system, all tester strains were also run, in triplicate, 

with known positive response chemicals. Positive controls not requiring metabolic activation 

were strain specific and were as follows:

TA1535 - without activation - Sodium azide (10 μg/plate)

TA1537 - without activation - 9-aminoacridine (150 μg/plate)

TA1538 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA98 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA100 - without activation - Sodium azide (10 μg/plate)

The positive control requiring metabolic activation was 2-anthramine (2-Aminoanthracene) (2.5 

μg/plate) in all strains.

Plate Incorporation Assay: Test article, 2,4-DETDA, Lot # 5712-38-3, was received as a clear 

yellow liquid and was dissolved in dimethylsulfoxide (DMSO) at the suggestion of the sponsor. 

The Preliminary Toxicity Screen was waived by the sponsor. The dose levels selected for the 

Plate Incorporation Mutation Assay in consultation with the sponsor were 625, 1250, 2500, 5000 

and 7500 μg/plate.

The test article, 2,4-DETDA, was evaluated in Ames/Salmonella tester strains TA1535, TR1537, 

TA1538, TA98 and TA100 with and without metabolic activation preparation. There was 0.10 

ml of S-9 supernatant (45 mg protein per ml) per 1.0 ml of S-9 mix in the rat liver metabolic 

activation preparation.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher lsotempR Dry 

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquot of top agar solution, 0.1 ml of tester 

strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation had, in addition to the 

preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats.

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

46

strain within an hour the plates were inverted and placed in a dark 37°C incubator. The plates 

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay, The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5nM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.

Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. 

RESULTS

Test article, 2,4-DETDA, Lot # 5712-38-7, did not induce a mutational dose response in 

Ames/Salmonella tester strains TA1535, TA1537, TA1538, TA98 and TA100 with and without 

metabolic activation preparation over the dose range of 625-7500 μg/plate. 

Mutant frequency values that were 2-fold the mean solvent control values were observed for 

strain TA100 with metabolic activation preparation at dose levels of 625 and 1250 μg/plate. 

However, due to insufficient test article this observation or the occurrence of a dose response at 

lower concentrations could not be confirmed. 

Genotoxicity result: Equivocal

Cytotoxic concentration (if applicable): N/A

Results remarks: All solvent and positive controls employed in the evaluation of the test article 

induced mutant frequency values that were within the acceptable range of mean historical values.

CONCLUSIONS

The test substance did not induce a mutational dose response in Salmonella strains TA1535, 

TAl537, TA1538, TA98 or TA100 with or without metabolic activation. Mutant frequency 

values that were 2-fold the mean solvent control values were observed for strain TA100 with 

metabolic activation preparation at dose levels of 625 and 1250 μg/plate. However, due to

insufficient test article this observation or the occurrence of a dose response at lower 

concentrations could not be confirmed. 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

47

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study and included 

cytotoxicity of the test substance.

REFERENCES

Barfknecht, T., Naismith, R., and Matthews, R. 1985b. Ames Salmonella/Microsome Plate Test 

with 2,4 DETDA Lot #5712-38-7 (EPA/OECD). Report # PH 301-ET-0 13-85. Performing 

Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

48

Study 5: Mutagenicity studies in Salmonella typhimurium (Barfknecht et al., 1985c)

Title: Ames Salmonella/microsome plate test (EPA/OECD)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: 99.51% 3,5-Diethyltoluene-2,6-diamine, CAS No. 2095-01-4; Synonym: 2,6-DETDA

Remarks: Lot No. 5712-53-73; Light brown solid; Composition: 99.51% 3,5-diethyltoluene2,6-diamine, 0.49% other trialkylates (as cited in Mulholland, 1986). 

METHOD

Method/guideline followed: Protocol-301 84 (EPA/OECD)

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1985

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 625, 1250, 2500, 5000 and 7500 μg/plate

Statistical methods used: In most tests with the Salmonella/microsome Assay, results are either 

clearly positive or clearly negative, A positive result is defined as a reproducible, dose-related 

increase in the number of histidine-independent colonies with at least one dose point inducing a 

mutant frequency valve that is two-fold the solvent control value. Significance at the 95% 

confidence Limit is determined by the program developed by Moore and Felton (1983). This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 

the 95% confidence limits of the historical mean (See Historical Data) for control values and the 

test chemical produces the highest increase equal to or greater than three times the solvent 

control value, the test chemical is considered positive. A negative result is defined as the absence 

of a reproducible increase in the number of histidine-independent colonies.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

49

Test method/conditions remarks:

Negative and Positive Controls: Tester strains TA1535, TA1537, TA1538, TA98 and TA100 

were plated in triplicate with the appropriate solvent, both with and without metabolic activation, 

to obtain background lawn and revertant colony formation to serve as negative solvent controls. 

In order to validate the integrity of the test system, all tester strains were also run, in triplicate, 

with known positive response chemicals. Positive controls not requiring metabolic activation 

were strain specific and were as follows:

TA1535 - without activation - Sodium azide (10 μg/plate)

TA1537 - without activation - 9-aminoacridine (150 μg/plate)

TA1538 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA98 - without activation - 2-nitrofluorene (5.0 μg/plate)

TA100 - without activation - Sodium azide (10 μg/plate)

The positive control requiring metabolic activation was 2-anthramine (2-Aminoanthracene) (2.5 

μg/plate) in all strains.

Plate Incorporation Assay: Test article, 2,6-DETDA, Lot # 5712-53-73, was received as a light 

brown solid and was dissolved in dimethylsulfoxide (DMSO) at the suggestion of the sponsor. 

The Preliminary Toxicity Screen was waived by the sponsor. The dose levels selected for the 

Plate Incorporation Mutation Assay in consultation with the sponsor were 625, 1250, 2500, 5000 

and 7500 μg/plate.

The test article, 2,6-DETDA, was evaluated in Ames/Salmonella tester strains TA1535, TA1537, 

TA1538, TA98 and TA100 with and without metabolic activation preparation. There was 0.10 

ml of S-9 supernatant (45 mg protein per ml) per 1.0 ml of S-9 mix in the rat liver metabolic 

activation preparation.

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher lsotempR Dry 

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquot of top agar solution, 0.1 ml of tester 

strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation had, in addition to the 

preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats.

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

50

strain within an hour the plates were inverted and placed in a dark 37°C incubator. The plates 

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay, The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5nM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.

Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. 

RESULTS

No increases in mutant frequency were observed in Ames/Salmonella test strains TA1535, 

TA1537, TA1538, TA98 and TA100 without metabolic activation preparation over the dose 

range of 625-7500 μg/plate. Results were also negative for strain TA1535 with metabolic 

activation preparation over the same dose range. Statistically significant (at the 95% confidence 

interval) linear mutational dose responses were observed for strains TA1537, TA1538 and TA98 

in the presence of metabolic activation over the dose range of 625-7500 μg/plate. A non-linear 

dose response was observed for strain TA100 with metabolic activation preparation over the 

same dose range. Maximum observed fold increases over mean solvent control mutant frequency 

values were as follows: TA1537 (7.2), TA1538 (33), TA98 (18.9) and TA100 (7.2). Due to 

insufficient test article these positive responses could not be confirmed. 

Genotoxicity result: Positive with metabolic activation

Cytotoxic concentration (if applicable): N/A

Results remarks: All solvent and positive controls employed in the evaluation of the test article 

induced mutant frequency values that were within the acceptable range of mean historical data.

CONCLUSIONS

The test substance was mutagenic in S. typhimurium strains TA1537, TA1538, TA98 and TA100 

in the presence but not the absence of metabolic activation.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

51

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study and included 

cytotoxicity of the test substance.

REFERENCES

Barfknecht, T., Naismith, R., and Matthews, R. 1985c. Ames Salmonella/Microsome Plate Test 

with 2,6 DETDA Lot #5712-53-73 (EPA/OECD). Report # PH 301-ET-014-85. Performing 

Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

52

Study 6: Mutagenicity studies in Salmonella typhimurium (Stewart and Riccio, 1985)

Title: In vitro microbiological mutagenicity assays of Ethacure 100/salicylic acid

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Ethacure 100/salicylic acid mixture (22:1); Brown, viscous liquid; Lot No.: Ethacure 

100:336, Salicylic acid: MCB, CB664. 

METHOD

Method/guideline followed: Not indicated

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1985

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 0 (DMSO solvent control), 10, 50, 100, 500, 1000 and 5000 μg/plate

Statistical methods used: No statistical analysis performed. Results are a tabulation of the 

number of colonies appearing on the plates.

Test method/conditions remarks: At the request of Sandia National laboratories, SRI 

International examined an Ethacure 100/salicylic acid mixture (22:1) for mutagenic activity in 

the Ames Salmonella/microsome assay. The assays were conducted with Salmonella

typhimurium tester strains TA1535, TA1537, TA1538, TA98, and TA100, both in the presence 

and absence of an Aroclor 1254-induced rat-liver metabolic activation system. 

Dimethylsulfoxide (DMSO) was used as the diluent.

After consultation with the client, the preliminary (range-finding) Assay was waived because this 

sample is similar to samples assayed previously. The first assay of this mixture was conducted at 

dose levels used in the previous work: 10, 50, 100, 500, 1000, and 5000 μg/plate.

Metabolic Activation: Aroclor 1254-induced, rat-liver S-9; SRI Batch F-6; ~ 31.9 mg/ml protein; 

Animal Supplier: Simonsen Laboratories, Gilroy, CA
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

53

Negative (Diluent) Control Material: Dimethylsulfoxide (DMSO)

Positive Control Chemicals: 9-Aminoacridine, 2-Anthramine, 2-Nitrofluorene and Sodium Azide

Criteria for Interpretation:

Positive: A test article is considered a mutagen when it produces a reproducible, dose-related 

increase in the number of revertants in one or more strains. This increase should occur for at least 

three dose levels.

Negative: A test article is considered a nonmutagen when no dose-related increase in the number 

of revertants is observed in at least two independent experiments. The maximum dose level 

tested for nontoxic compounds is 10 mg/plate (unless dictated otherwise by the Sponsor or by 

solubility problems). For toxic compounds, only the highest dose level tested should show 

evidence of toxicity.

Inconclusive: When a test article cannot be identified clearly as a mutagen or nonmutagen in the 

standard plate assay, the results are classified as inconclusive.

RESULTS

The results of this assay showed significant increases in the number of his

+ revertant colonies 

with strains TA1538, TA98, and TA100 in the presence of metabolic activation. Because these 

increases occurred in and/or near dose levels that exhibited toxicity, colonies were replicated or 

restreaked onto minimal plates lacking histidine, but supplemented with biotin, to check for their 

his+ characteristics. These colonies were found to be true revertants and not artifacts due to 

toxicity. Slightly increased numbers of revertants were also seen in this assay with TA1537 in 

the presence of metabolic activation. Toxicity indicated by a decrease in the number of revertants 

with or without concurrent thinning of the background growth lam-was seen at the highest dose 

level with several of the tester strains.

The second assay of the Ethacure 100/salicylic acid mixture without metabolic activation was 

conducted at the same dose levels as in the first assay. The dose levels used in the presence of 

metabolic activation were slightly higher (except for TA100) and covered a narrower range, as 

necessary to define a better dose response. Again, no mutagenic activity was seen in the absence 

of metabolic activation, and toxicity was seen with several of the tester strains at the highest one 

or two dose levels. Significant, clearly dose-related increases in the number of revertant colonies 

occurred with strains TA1538, TA98, and TA100, but not with strain TA1537. Because the 

previously increased values seen with TA1537 were so slight and not reproducible, we attributed 

them to random fluctuations in the number of spontaneous revertants and not to mutagenic

activity.

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): 5000 μg/plate
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

54

Results remarks: Positive and negative (diluent) control values were within acceptable limits 

for all assays.

CONCLUSIONS

Ethacure 100/salicylic acid mixture (22:1) was mutagenic in S. typhimurium strains TA1538, 

TA98 and TA100 in the presence but not the absence of metabolic activation. The test substance 

as not mutagenic in S. typhimurium strains TA1535 or TA1537 with or without metabolic 

activation.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study provided and 

included purity and composition of the test substances, GLP compliance and test 

method/guidelines followed.

REFERENCES

Stewart, K., and Riccio, E. 1985. In Vitro Microbiological Mutagenicity Assays of Ethacure 

100/Salicylic Acid. Performing Laboratory: SRI International, Menlo Park, CA. Sponsor:

Sandia National Laboratories.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

55

Study 7: Mutagenicity studies in Salmonella typhimurium (Johnson and Pettigrew, 1987a)

Title: Salmonella/microsomal assay with DETDA (Lot #5868-1)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear brown liquid; Lot #5868-1. 

METHOD

Method/guideline followed: Protocol - Ettox 027

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

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

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 0 (untreated control), 0 (DMSO solvent control), 10, 37.5, 75, 100 and 

125 mg/mL

Statistical methods used: Not indicated

Test method/conditions remarks: Salmonella typhimurium strains TAl535, TA1537, TA98 

and TA100 were treated in the presence of metabolic activation (Aroclor 1254 induced rat liver 

S-9 fraction) with DETDA in DMSO. Dose levels tested were 125, 100, 75, 37.5, and 10 mg/mL. 

DETDA was soluble at all dose levels tested. Each dose was treated in triplicate. An untreated 

control, solvent control and positive control were treated concurrently.

RESULTS

125 mg/mL demonstrated toxicity in all strains tested with metabolic activation. DETDA caused 

a dose related increase in mutant colonies in strains TAl535, TA1537, TA98, and TA100 in the 

presence of metabolic activation.

Genotoxicity result: Positive
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

56

Cytotoxic concentration (if applicable): 125 mg/mL in all strains tested with metabolic 

activation

Results remarks: Color and appearance of the test article did not change from the time of 

receipt to the time of use.

CONCLUSIONS

DETDA was genetically active in the Salmonella/ Microsomal Assay according to Protocol -

Ettox 027.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study summary 

provided and included purity and composition of the test substance and statistical methods used.

REFERENCES

Johnson, D., and Pettigrew, A. 1987a. Genetic toxicology. Salmonella/Microsomal Assay with 

DETDA (Lot #5868-1). Ames 101 - #099. Sponsor: Ethyl Corporation, Baton Rouge, LA.
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

57

Study 8: Mutagenicity studies in Salmonella typhimurium (Jagannath and Brusick, 1977)

Title: Mutagenicity evaluation of DEDTA (diethyltoluenediamine) - final report

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Dark amber liquid, referred to as “DETA”, identified as “diethyltoluenediamine”. 

METHOD

Method/guideline followed: Not indicated

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1977

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

Saccharomyces cerevisiae strain D4 

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

adult human male livers)

Concentrations tested: 0 (DMSO solvent control), 0.001, 0.01, 0.1, 1 and 5 μl/plate

Statistical methods used: Not indicated

Test method/conditions remarks: The test compound was examined for mutagenic activity in 

a series of in vitro microbial assays employing Salmonella and Saccharomyces indicator 

organisms. The compound was tested directly and in the presence of liver microsomal enzyme 

preparations from human liver.

Positive controls in the non-activation assay were methylnitrosoguanidine (MNNG) for strains 

TA1535, TA100 and D4 (10 μg/plate), 2-nitrofluorene (NF) for strains TA1538 and TA98 (100 

μg/plate) and quinacrine mustard (QM) for strain TA1537 (10 μg/plate). Positive controls in the 

activation assay were 2-anthramine (ANTH) for strains TA1535 and TA100 (100 μg/plate), 2-

acetylaminofluorene (AAF) for strains TA1538 and TA98 (100 μg/plate) and 8-aminoquinoline 

(AMQ) for strain TA1537 (100 μg/plate).

Either deionized water or dimethylsulfoxide (DMSO) was used to prepare stock solutions of 

solid materials. All dilutions of the test materials were made in either deionized water or DMSO. 
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Plate Test (Overlay Method): Approximately 108 cells from an overnight culture of each 

indicator strain were added to separate test tubes containing 2.0 ml of molten agar supplemented 

with biotin and a trace of histidine. For nonactivation tests, at least four dose levels of the test 

compound were added to the contents of the appropriate tubes and poured over the surfaces of 

selective agar plates. In activation tests, a minimum of four different concentrations of the test 

chemical were added to the appropriate tubes with cells. Just prior to pouring, an aliquot of 

reaction mixture (0.5 ml containing the 9,000 x g liver homogenate) was added to each of the 

activation overlay tubes, which were then mixed, and the contents poured over the surface of a 

minimal agar plate and allowed to solidify. The plates were incubated for 48 hours at 37°C, and 

scored for the number of colonies growing on each plate. Positive and solvent controls using 

both directly active positive chemicals and those that require metabolic activation were run with 

each assay.

The numbers of colonies on each plate were counted and recorded on printed forms. These raw 

data were analyzed in a computer program and reported on a printout. The results are presented 

as revertants per plate for each indicator strain employed in the assay. The positive and the 

solvent controls are provided as reference points. Other relevant data are provided on the 

computer printout.

RESULTS

The compound was tested over a series of concentrations such that there was either quantitative 

or qualitative evidence of some chemically-induced physiological effects at the high dose level. 

The low dose in all cases was below a concentration that demonstrated any toxic effect. The dose 

range employed for the evaluation of this compound was from 0.001 μl to 5 μl per plate. The 

compound was slightly toxic to the strain TA-1535 and TA-1537 at 5 μl per pt ate.

The results of the tests conducted on the compound in the presence and absence of the human 

liver activation system were all negative.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 5 μl/plate for TA1535 and TA1537

Results remarks: None indicated

CONCLUSIONS

The test compound, DETDA, did not demonstrate mutagenic activity in any of the assays 

conducted in this evaluation and was considered as not mutagenic under these test conditions.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study summary 

provided and included purity and composition of the test substances, GLP compliance, test 

method/guidelines followed and statistical methods used.
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REFERENCES

Jagannath, D., and Brusick, D. 1977. Mutagenicity Evaluation of DETA 

(Diethyltoluenediamine) Final Report. Performing Laboratory: Litton Bionetics, Kensington, 

MD. Sponsor: Ethyl Corporation.
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Study 9: Mutagenicity studies in Salmonella typhimurium (Barfknecht et al., 1985d)

Title: Ames Salmonella/microsome plate test (EPA/OECD)

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Lot # 5711-75-1 (Orangeburg sample); Light brown liquid; Composition: 0.962% 

METDA, 78.228% 2,4-DETDA, 19.740% 2,6-DETDA, 0.251% 2,4,6-TETDA, 0.595% 

trialkylated PDA, 0.225% other (as cited in Mulholland, 1986).

METHOD

Method/guideline followed: Not indicated

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1985

Test species: Salmonella typhimurium strains TA1538, TA98 and TA100 

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 100, 333, 1000, 3333 and 10,000 μg/plate

Statistical methods used: In most tests with the Salmonella/microsome Assay, results are either 

clearly positive or clearly negative, A positive result is defined as a reproducible, dose-related 

increase in the number of histidine-independent colonies with at least one dose point inducing a 

mutant frequency valve that is two-fold the solvent control value. Significance at the 95% 

confidence Limit is determined by the program developed by Moore and Felton (1983). This 

program applies a linear regression analysis to the data points and any P value greater than 0.05 

is considered significant. In addition, the greater the P value, the higher the probability that the 

data points fit a linear response. This dose-response relationship occasionally necessitates slight 

modification of the original doses in a repeat assay. Alternatively, if the solvent control is within 

the 95% confidence limits of the historical mean (See Historical Data) for control values and the 

test chemical produces the highest increase equal to or greater than three times the solvent 

control value, the test chemical is considered positive. A negative result is defined as the absence 

of a reproducible increase in the number of histidine-independent colonies.

Test method/conditions remarks: Test article, 5711-75-1, was received as a light brown 

liquid and was dissolved in dimethylsulfoxide (DMSO) at the suggestion of the sponsor. The 
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dose levels evaluated in the Ames/Salmonella Mutation Assay with tester strains TA1538, TA98 

and TA100 in the presence of metabolic activation preparation were 100, 333, 1000, 3333 and 

10,000 μg/plate. There was 0.00 ml of S-9 supernatant (35.7 mg protein per ml) per 1.0 ml of rat 

liver metabolic activation preparation. Plates were scored after approximately 48 hours and 72 

hours of incubation at 37°C ± 2°C.

Tester strains TA1538, TA98 and TA100 were plated in triplicate with the appropriate solvent, 

with metabolic activation, to obtain background lawn and revertant colony formation to serve as 

negative solvent controls. In order to validate the integrity of the test system, all tester strains 

were also run with known positive response chemicals, six (6) plates being seeded per point.

The positive control requiring metabolic activation was 2-anthramine (2-Aminoanthracene) (2.5 

μg/plate) in all strains. 

Top Agar: Top agar, used as an overlay, was reconstituted into a molten state and supplemented 

with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 

45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher lsotempR Dry 

Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All

compound-treated tubes and plates were done in triplicate. Using sterile technique, the following 

were added to each tube in the following order: 2 ml aliquot of top agar solution, 0.1 ml of tester 

strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were 

vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the 

plate, and the top agar overlay was allowed to harden.

Metabolic Activation System: Tubes requiring metabolic activation had, in addition to the 

preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 

1254-treated Sprague Dawley rats.

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was 

added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates 

were allowed to harden for several minutes. The same procedure was repeated for each tester 

strain within an hour the plates were inverted and placed in a dark 37°C incubator. The plates 

were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting 

revertant colonies on an electronic colony counter interfaced with an Apple computer for data 

acquisition.

Bacterial Contaminant Control: To insure the quality of aseptic technique during the assay and 

also the sterility of solvents, compounds and equipment, standard contamination checks were 

conducted with the assay. These contamination checks included the top level of test substance, 

solvent, top agar and S-9 mix at the same volumes as in the assay, The test substance or S-9 mix 

were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and 

poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. 

All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then 

scored for contamination.
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Data Reporting: In scoring the assay, the positive and negative controls were first evaluated. If 

the negative control values did not fall within the acceptable historical mean values, the 

remaining plates were not scored and the assay was repeated. 

RESULTS

The results for test article 5711-75-1 were positive in Ames/Salmonella tester strains TA1538, 

TA98 and TA100 with metabolic activation preparation over the dose range of 100-10,000 

μg/plate. These findings are based on the observation of reproducible, statistically significant, 

linear mutational dose responses after 48 and 72 hours of incubation. In addition, maximum fold

increase mutant frequency values which were observed a t the 48 hour incubation period were 

19.7, 22.8 and 3.4 for strains TA1538, TA98 and TA100, respectively. 

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not indicated

Results remarks: All solvent and positive controls employed in the evaluation of the test article 

induced mutant frequency values that were within the acceptable range of mean historical data 

except the solvent control value for strain TA98 was outside of the acceptable limits after the 72 

hour incubation period. The sponsor requested that the laboratory not repeat strain TA98.

CONCLUSIONS

Test article, 5711-75-1, induced a positive mutational response in Ames\Salmonella tester strains 

TA1538, TA98 and TA100 in the presence of metabolic activation preparation over the dose 

range of 100-10,000 μg/plate under the conditions employed for the study. The data suggest that 

the test article induces frame shift and base-pair change mutational events in the 

Ames/Salmonella tester strains.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study summary 

provided and included test method/guidelines followed and cytotoxicity of test substance.

REFERENCES

Barfknecht, T., Naismith, R., and Matthews, R. 1985d. Ames Salmonella/Microsome Plate Test: 

5711-75-1 (EPA/OECD). Report # PH 301-ET-009-85. Performing Laboratory: Pharmakon 

Research International, Waverly, PA.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter-Office Memo. 

Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 10: Mutagenicity studies in Salmonella typhimurium (Stankowski and Naismith, 

1987a)

Title: Ames/Salmonella Plate Incorporation Assay with DETDA Samples 544-dist, 663,692, 

705 and 722

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: DETDA Samples 544-dist, 663,692, 705, 722 and Lot #565-8; dark red (544-dist, 

663, 692 and 705) or yellow/amber (722) liquids. 

METHOD

Method/guideline followed: Protocol- 301

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

Test species: Salmonella typhimurium strains TA1538, TA98 and TA100 

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 

Assay 1: 1000, 3750, 7500, 10,000 and 12,500 μg/plate

Assay 2: 500, 1000, 3750, 7500, 10,000 and 12,500 μg/plate

Assay 3: 100, 250, 500, 1000, 2500, 3750 and 5000 μg/plate

Statistical methods used: A positive result is defined as a reproducible, statistically significant 

two-fold dose-dependent increase in the number of histidine-independent colonies, with at least 

one dose point inducing an average revertant frequency that is two-fold that of the solvent 

control. Significance is determined using the program developed by Snee and Irr (1981). This 

program applies a linear regression analysis to the data points and any p value less than 0.05 is 

considered significant. Alternatively, if the test article produces an increase greater than or equal 

to three times the solvent control value (in the absence of a dose-dependent increase), the test 

chemical is considered positive. A negative result is defined as the absence of a dose-dependent 

two-fold increase in the number of histidine-independent revertants (or three-fold increase in the 

absence of any dose-dependency).
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Test method/conditions remarks: Five DETDA samples (Lot #s 544-dist, 663, 692, 705 and 

722) were evaluated in the Ames/Salmonella Plate Incorporation Assay to determine their ability 

to induce reverse mutations at selected histidine loci in three tester strains of Salmonella

typhimurium in the presence of an exogenous metabolic activation system (S9). The five 

samples were evaluated in triplicate cultures in strains TA1538, TA98 and TA100 in the 

presence of S9 at doses of 1000, 3750, 7500, 10,000 and 12,500 μg/plate. These doses were 

selected by the sponsor based upon results for other DETDA samples previously evaluated.

In addition, a sixth DETDA sample (Lot #565-8) was also evaluated concurrently as a positive 

control at a single dose of 7500 μg/plate with S9. The S9 mixture included 6% (v/v) Aroclor 

1254-induced male Sprague-Dawley rat liver homogenate with the appropriate buffer and 

cofactors. 

Salmonella which have undergone reversion to his+ form colonies in the absence of histidine. In 

contrast, his- Salmonella can only undergo a limited number of doublings (due to the histidine 

supplement in the top agar) and form the typical background lawn. Following incubation for 48 

hours, revertant colonies are enumerated with an automated colony counter. All mutation assays

are performed in triplicate cultures in all three tester strains for each test article dose, as well as 

positive and solvent centrals. All five DETDA samples were evaluated at doses of 1000, 3750, 

7500, l0,000 and 12,500 μg/plate in the presence of S9. However, toxicity was observed for all 

five DETDA samples in all three strains at doses of 7500, 10,000 and/or 12,500 μg/plate. In 

addition, approximately 1.6- to 3.3-fold increases in revertant frequencies were observed for the 

test samples in strain TA100. Therefore, all five samples were re-evaluated in all three strains at 

doses of 500, 1000, 37 50, 7500, 10,000 and 12,500 μg/plate with S9, DETDA sample 544-dist 

was subsequently re-evaluated in a third assay in all three strains at doses of 100, 250, 500, 1000, 

2500, 3750 and 5000 μg/plate with S9.

Treatment with Test and Control Articles: Treatments were performed by combining 2 ml top 

agar (supplemented with 0.5mM histidine/0.5mM biotin), 0.1 ml tester strain, 0.5 ml of the S9 

mixture and 0.1 ml test or control article or solvent in sterile glass tubes preheated to 45°C. The 

S9 mixture contained 8mM MgCl2, 33mM KC1, 4mM NADP, 5mM glucose-6-phosphate, 

100mM NaH2 PO4 (pH 7.4) and 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver 

homogenate. The tubes were vortexed end the mixture was poured onto minimal glucose plates, 

evenly distributed, and allowed to solidify. Within an hour the plates were inverted and 

incubated in the dark at 37°C for 48 hours.

Bacterial Contaminant Evaluation: To ensure the sterility of solvents, compounds and 

equipment, standard contamination evaluations were performed with the assay. The solvent, top

agar, S9 mix, and top dose of the test article were evaluated at the same volumes used in the 

assay. The test article, solvent and S9 mix were evaluated as in the mutation assay, but in the 

absence of added Salmonella. Top agar alone was also plated on minimal glucose plates. All

plating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for 

bacterial growth.

Scoring: Following the 48-hour incubation, the background lawn and spontaneous revertants 

were scored for normal, inhibited or no growth. Inhibited growth was characterized by the 

absence of a confluent bacterial lawn and/or the presence of pindot colonies. Revertant colonies 
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65

were enumerated on an Artek electronic colony counter interfaced with an Apple computer for 

data acquisition. Solvent and positive controls were scored first, and test article treated cultures 

were scored only if the average negative control values were within historical ranges.

RESULTS

Revertant frequencies for all doses of all five samples in strains TA1538 and TA98 approximated 

or were less than those observed in the concurrent negative control cultures. In contrast, 

statistically significant increases in revertant frequencies of approximately 1.6- to 3.3-fold 

background were observed for samples 544-dist, 663, 692, 705 and 722 in strain TA100 (p< 

0.05). The maximal increases observed were generally limited to the range of 1000-7530 

μg/plate; higher doses produced lower revertant frequencies due to toxicity.

Therefore, all five DETDA samples were re-evaluated at doses of 500, 1000, 3750, 7500, 10,000 

and 12,500 μg/plate with S9. Inhibited growth was again observed for all five samples in all 

strains at doses of 7500, 10,000 and/or 12,500 μg/plate. Revertant frequencies for all doses of 

DETDA 544-dist, 663, 692, 705 and 722 in strains TA1538 and TA98 approximated or were less 

than those of the concurrent negative control cultures. However, statistically significant increases 

in revertant frequencies of approximately 1.3-, 1.8-, 1.6-, 1.7- and 1.7-fold control values were 

again observed in strain TA100 for samples 544-dist, 663, 692, 705 and 722, respectively (p<

0.05). In addition, these increases appeared to be dose-dependent over the range of 500-3750 

μg/plate for samples 663, 692, 705 and 722. DETDA-544 (dist) was re-evaluated in a third assay 

in all three strains at doses of 100, 250, 500, 1000, 2500, 3750 and 5000 μg/plate with S9. In this 

final assay, a statistically significant increase in revertant frequency, equal to 1.3 – fold

background, was observed in strain TA100 only at a dose of 500 μg/plate. 

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): ≥ 7500 μg/plate

Results remarks: All positive and negative control values in both assays were within 

acceptable limits.

CONCLUSIONS

The results for DETDA samples 663, 692, 705 and 722 were positive in the Ames/Salmonella

Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.

Although DETDA 544-dist reproducibly induced statistically significant increases in revertant 

frequencies in all three experiments, none of the increases were 2-fold background and DETDA 

544-dist is therefore considered to be negative.

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study provided and 

included purity and composition of the test substance.
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REFERENCES

Stankowski, L., and Naismith, R. 1987a. Ames/Salmonella Plate Incorporation Assay with 

DETDA Samples 544-dist, 663,692, 705 and 722. Report # PH 301-ET-002-87. Performing 

Laboratory: Pharmakon Research International, Waverly, PA.
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Study 11: Mutagenicity study in Salmonella typhimurium (Schechtman et al., 1978)

Title: Activity of DETDA in the Salmonella/microsomal assay for bacterial mutagenicity

This study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Test substance referred to as “DETA”, identified as “diethyltoluenediamine”, brown 

liquid

METHOD

Method/guideline followed: The test was performed according to the method of Ames et al.

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1978

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

Sprague-Dawley adult male rat livers)

Concentrations tested: 0 (DMSO solvent control), 0 (untreated control), 0.05, 0.1, 0.5, 1 and 5 

μl/plate

Statistical methods used: Linear regression analysis to determine strength of dose response

Test method/conditions remarks: For the purposes of this study, DETDA was used as a 5.0% 

solution (v/v) in DMSO.

Mutagenesis Assay without Metabolic Activation: 

The test was performed according the method of Ames et al. For the assay, the following were 

added, in order, to 16 x 125 mm pre-capped tubes:

2 ml molten agar (0.6% Noble’s agar supplemented with 0.6% NaCl, 0.5 mM biotin, and 0.5 mM 

histidine). 

0.1 ml of bacterial culture

0.1 ml of test agent in the appropriate solvent. 
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Tubes were mixed quickly but gently and the contents poured over a base plate of Spizzizen’s 

minimal medium. Plates were rotated to distribute the overlay agar, allowed to harden and 

incubated at 37C for 48 hr at which time the number of mutant colonies were determined. 

Each agent was tested at five concentrations against all five strains of bacteria. 

Three plates were made for each control and each dilution of test agent. The following controls 

were included in each experiment. 

Bacteria only for spontaneous reversion

Bacteria and solvent for solvent effect

Known positive mutagens: 

TA1535: N-methyl-N-nitro-N-nitrosoguanidine, 5 μg/plate

TA100: N-methyl-N-nitro-N-nitrosoguanidine, 5 μg/plate

TA1537: 9-Aminoacridine, 100 μg/plate

TA1538: 2-Nitrofluorene, 5 μg/plate

TA98: 2-Nitrofluorene, 5 μg/plate

Sterility

The solvent and each dilution of test agent were checked for sterility by adding 0.1 ml of each to 

2 ml overlay agar without bacteria and pouring over a minimal medium base plate. Plates were 

incubated as above. 

The solvent was determined by the solubility of the test agent. Whenever possible, distilled 

water was the solvent of choice. DMSO, ethanol, acetone and ethyl acetate may also be used in 

descending order of preferences. The compound tested in this study was dissolved in DMSO.

Non-specific induction of liver enzymes: Aroclor 1254, a polychlorinated biphenyl, was used for 

the non-specific induction of liver enzymes in rats. Adult male Sprague-Dawley rats, weighing 

200-225 g, were given a single IP injection of 500 mg/kg Aroclor 1254 in corn oil. On day 5 post 

injection, animals were sacrificed by CO2 asphyxiation and the livers were removed and the S-9

fraction prepared. Animals had free access to food until12 to 15 hr before sacrifice; water was 

available at all times.

Preparation of S-9 fraction of the liver homogenate: All operations were performed aseptically at 

0-4°C using sterile solutions and glassware. After surgical removal and weighing, livers were 

minced in three volumes 0.15 M KC1 and homogenized in a Potter Elvejhem tissue grinder at 

120-150 rpm. The homogenate was centrifuged at 9900 X g for 20 min and the supernatant 

decanted and saved and designated as the S-9 fraction, The S-9 was divided into 1 ml amounts 

and stored frozen at -80°C until the day of use.

Preparation of S-9 mix: On the day of the assay, sufficient S-9 for that day's use was thawed and 

mixed with cofactors for use in the assay. S-9 mix contains per ml: S-9 (generally 50 μl to 100 

μl), 0.8 MgCl2 , 0.33 mM KCl, 0.5mM glucose 6 phosphate, 0.4 RM NADP, 2and 100 mM 

NaH2PO4 *H2O, pH 7.4. Reagents for the S-9 mix were weighed and prepared fresh daily with 

the exception of sodium phosphate which was made as a stock solution of 0.2 M. The entire S-9 
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mix was filter sterilized before use through a 0.45 u Nalgene disposable filter unit. S-9 mix was 

kept at 0-4°C until use; unused portions were discarded at the end of the day.

Standardization of S-9 for use in the assay: Each bulk preparation of S-9 was assayed for aryl

hydrocarbon hydroxylase (AHH) activity; protein was determined according to the method of 

Weigele et al.

Prior to use in the assay, each S-9 preparation was titrated over a range of concentrations (10-300 

μl S-9 per ml of S-9 mix) with 3-methylcholanthrene (3MC) , benzo(a)-pyrene (BP) and 6-

aminochrysene (6-AC) to determine the optimum amount of S-9 for use in a general screening

procedure. This was usually in the range of 50 to 100 μl per ml of S-9 mix.

The activity of the S-9 preparation was confirmed by its ability to activate the control mutagens, 

2-AA and Aflatoxin B1 which require metabolism to exert their effect, to reactive derivatives.

Mutagenesis Assay with Metabolic Activation:

The assay was the same as that in the non-metabolic activation assay described above except that 

0.5 ml of S-9 mix was added to the overlay agar immediately before it was mixed and poured. 

Plates were incubated and scored as noted above. 

Controls for the assay with metabolic activation were:

Bacteria with S-9 mix to determine the effect of S-9 upon spontaneous reversion

Bacteria, S-9 mix and solvent to determine the effect of the solvent

Positive control mutagens: 

TA1535: 5 μg 2-Aminoanthracene

TA100: 1 μg Aflatoxin B1

TA1537: 1 μg 6-Aminochrysene

TA1538: 2 μg 2-Aminofluorene

TA98: 1 μg Aflatoxin B1

Sterility controls on each dilution of a test agent were done as noted above. One-half ml of the 

S-9 mix with neither test agent nor bacteria was added to 2 ml overlay agar and poured on to a 

minimal medium base plate. Plate sterilities were also performed. 

Before an agent is reported to be either active or inactive in the Salmonella/microsomal assay, 

the following criteria must be met:

All sterility controls must be negative for bacterial growth 

The spontaneous revertants for each strain must be within acceptable limits.

All 3 control plates must have approximately equal numbers of revertant colonies. The solvent 

controls must have approximately the same number of colonies as spontaneous reversion 

controls. Positive mutagens must give at least 3x the number of colonies as the controls for

spontaneous reversion. Any test with a strain which does not meet these criteria must be repeated 

on a separate day.
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70

All criteria noted above must be met before results with an unknown agent can be evaluated. To 

be considered positive, an unknown agent should exhibit a dose response effect. That is, there 

should be an increasing number of mutants with increased amounts of test agent. For strains

TA1535, TA1537, TA1538 and TA98, the first positive dose must produce 3x the number of 

colonies as the negative control. For strain TA100, at least one dose tested must produce 3.5x the 

number of mutants as the spontaneous reversion control.

RESULTS

The test compound without activation did not induce a significant increase in the number of 

revertant colonies over that shown in the untreated control plates in strains TA1538, TA100 and 

TA98.

The test compound without activation did induce a significant increase in the number of revertant 

colonies over that seen in the untreated control plates for TA1535 and TA1537.

The test was also performed with the addition of an exogenous source of liver enzymes. This did 

not affect the activity of the test agent in this system for strains TA1535, TA1537, TAl538, 

TA100 and TA98.

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Toxicity was not observed

Results remarks: Negative and positive controls responded appropriately.

CONCLUSIONS

The test agent did not induce a significant increase in the number of point mutations in 

Salmonella typhimurium strains in the absence of the activating system for strains TA1538,

TA100 and TA98. 

The test agent did induce a significant increase in the number of point mutations in Salmonella 

typhimurium strains in the absence of the activating enzyme system for strains TA1535 and

TA1537.

It also did not induce a significant increase in the number of point mutations with the addition of 

an exogenous source of liver enzymes for metabolic activation in strains TA1535, TA1537,

TA1538, TA100 and TA98.

STUDY RELIABILITY

[2] Reliable with restrictions. Important details were missing from the study including purity 

and composition of the test substance and GLP compliance.
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REFERENCES

Schechtman, L., Parmar, A., Fabrizio, D. 1978. Activity of DETA in the 

Salmonella/Microsomal Assay for Bacterial Mutagenicity. Final Report. Project No. T1360. 

Performing Laboratory: Microbiological Associates, Inc., Bethesda, MD. Sponsor: Ethyl 

Corporation, Baton Rouge, LA. 
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Study 12: Mutagenicity study - In vitro gene mutation in mammalian cells (Durward and 

Nolan, 2000)

Title: LZ1224.20: L5178Y TK +/- Mouse Lymphoma Assay

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonyms: 

Diethyltoluenediamine (DETDA), DETDA 80; Purity: 98.4%; Composition of test material: 

77.6% 3,5-diethyltoluene-2,4-diamine (GC), 20.8% 3,5-diethyltoluene-2,6-diamine (GC); 

Impurities: 0.4% ethyl toluenediamine (GC), < 0.0150% toluenediamine (GS), 0.02% (w/w) 

water

Remarks: Amber slightly viscous liquid; Batch No. 2235

METHOD

Method/guideline followed: OECD 476

Test type: In vitro mammalian cell gene mutation

GLP compliant?: Yes

Year study performed: 1999

Test species: Mouse lymphoma L5178Y cells 

Metabolic activation: With and without

Concentrations tested: 0 (negative control); 0 (DMSO vehicle control); Preliminary toxicity 

test: 28.1 to 1800 μg/mL; Experiment 1: 28.1 to 600 μg/mL (without S9), 225 to 1800 μg/mL 

(with S9); Experiment 2: 25 to 400 μg/mL (without S9), 200 to 1100 μg/mL (with S9); 

Experiment 3: 300 to 1300 μg/mL (with S9) 

Statistical methods used: The statistics were based on the UKEMS recommended procedures 

as reported by Cole et al. (1990).

For DETDA to have a ‘significant’ result, two or more of the following criteria had to be met:

1. A statistically significant increase in mutant frequency

2. A greater than threefold increase in the mutant frequency per survivor over the negative 

control value

3. A concentration-related increase in the mutant frequency per survivor

4. An increase in the absolute number of mutants

DETDA would be reported equivocal if only one of the above criteria was met.
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Test method/conditions remarks: 

A preliminary toxicity test was performed on cell cultures using a 3-hour exposure time both 

with and without metabolic activation (S9) and a continuous 24-hour exposure without S9. The 

cells were incubated at 37°C for 10 to14 days and then scored for colony formation. The plating 

efficiency values obtained were adjusted for each concentration level to account for immediate 

post treatment toxicity as a comparison to the vehicle control to give a percent survival value. A 

comparison of each percent survival value to the concurrent vehicle control was performed to 

give a relative survival value.

For mutagenicity tests, duplicate cultures (A and B) were established for each concentration level 

both with and without activation. 

Mutagenicity Tests

Experiment 1: Cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in 

sterile plastics universals. The treatments were performed with and without S9 at six 

concentration levels of DETDA, vehicle, or positive controls brought to a total volume of 20 mL. 

The treatment vessels were incubated at 37°C for 3 hours with continuous shaking using an 

orbital shaker.

Experiment 2: Cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in 

sterile plastics universals for the 3-hour pulse treatment with S9. In the absence of S9, the 

exposure period was continuous for 24 hours using 10 mL cultures of 0.3 x 106 cells/mL in 25 

cm

2 tissue culture flasks. After additions of S9, test material, or controls, the volume was 

brought to 20 mL and the treatment vessels were incubated at 37°C with continuous shaking 

using an orbital shaker. 

Experiment 3: In both Experiments 1 and 2, DETDA induced a small statistically significant 

concentration-related increase in the mutant frequency over the vehicle control. However, in 

experiment 1, there were only three concentration levels that were not too toxic, and in 

Experiment 2, optimum toxicity (10-20% survival) was not achieved. Therefore, a third 

confirmatory experiment was performed to investigate the reproducibility of the response. The 

test conditions used were the same as for the first two experiments.

At the end of the treatment period, for each experiment, the test media were removed by 

centrifugation and the cells were washed twice in R10 medium and resuspended in R20 medium 

at a cell density of 2 x 105 cells/mL. A sample of cells was removed for dilution to 10 cells/mL 

and plating in non-selective medium to determine Day 0 viability. The percent survival value 

was adjusted to account for immediate post-treatment toxicity as a comparison to the vehicle 

control using the cell count data. A comparison of each treatment percent survival value to the 

concurrent control value was performed to give a relative survival value. 

The remaining cells were incubated and subcultured daily for the required expression period, by 

counting and dilution to 2 x 105 cells/mL. On the second day after the end of the treatment 

period, the cells were counted, diluted to 104 cells/mL, and plated for mutant frequency (2000 

cells/well) in selective media containing 4 μg/mL 5-trifluorothymidine (5-TFT). After 10 to 14 

days of incubation, the plates were scored using a magnifying mirror box. To assist the scoring 
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of the TFT mutant colonies, MTT solution was added to each well of the mutation plates and the 

plates were incubated for approximately 2 hours. The number of positive wells (wells with 

colonies) and numbers of small and large colonies were recorded. The mutation frequency was 

calculated using the following equation:

Mutation Frequency per survivor = [(-lnP(0) selective medium)/cells per well in selective 

medium)]/surviving fraction in non-selective medium, where P(0) is the number of negative 

wells/total wells plated. 

Criteria for Determination of a Valid Test – The laboratory’s normal range for mutant frequency 

per survivor is 26-150 x 106 for the TK+/- locus in L5178Y cells. Vehicle control results should 

be within this range, although minor errors in cell counting and dilution or exposure to the 

metabolic activation system may cause this to be slightly elevated. Experiments where the 

vehicle control values are markedly greater than 200 x 106 mutant frequency per survivor are not 

normally acceptable and will be repeated. Positive controls should produce a significant (at least 

3- to 5-fold) increase in mutant frequency per survivor over the negative controls.

Vehicle Used?: Yes

Vehicle Name: DMSO

Positive Control Substances: Without metabolic activation: ethylmethanesulphonate 

(Experiments 1 and 2). With metabolic activation: cyclophosphamide (Experiments 1, 2, and 3). 

RESULTS 

Genotoxicity result: Positive

Cytotoxic concentrations (if applicable): Experiment 1: 1200, 1500, 1800 μg/mL (with S9), 

600 μg/mL (without S9); Experiment 2: 300, 400 μg/mL (without S9); Experiment 3: 1300 

μg/mL (with S9)

Results remarks: Preliminary Toxicity Test: In the presence of S9, toxicity was observed with 

DETDA at 1800 μg/mL. In the absence of S9, the toxicity was concentration-related, such that 

after a 3-hour exposure, toxicity was evident at and above 112.5 μg/mL. At the extended 

exposure period of 24 hours in the absence of S9, toxicity occurred at all concentrations, with a 

rapid onset of toxicity such that no cells were observed at and above 900 μg/mL. No test 

material precipitate was noted.

Mutagenicity Test

Experiment 1: The test material induced a concentration-related toxic response as indicated by 

the Day 0 viability values and relative survival values. There was also evidence of a reduction in 

Day 2 viability and relative total growth with the test material. The toxicity was such that in the 

absence of metabolic activation the maximum test material concentration level of 600 μg/mL, 

and in the presence of metabolic activation 1200, 1500, and 1800 μg/mL, were excluded from 

the statistical analysis because their toxicity exceeded the maximum acceptable level of 90%. 
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This resulted in the group with S9 having only three surviving concentration levels with 

acceptable toxicity, whereas, ideally there should have been four concentration levels. 

DETDA induced a statistically significant concentration-related (linear trend) increase in the 

mutant frequency at 106 per viable cell in the presence of S9 at and above 900 μg/mL. The 

increase was partly due to small colony formation indicating a clastogenic response. 

Experiment 2: The 24-hour continuous exposure without S9 treament demonstrated that the 

extended time point lowered the concentration level of the test material where the onset of 

toxicity occurred. As seen in Experiment 1, there was evidence of a concentration-related 

reduction in Day 0 viability and relative survival values in cultures treated with DETDA in the 

absence of S9. There was evidence of a reduction in Day 2 viability and relative total growth. 

The toxicity observed with the test material at 300 and 400 μg/mL was considered to be 

excessive, and therefore, these concentration levels were excluded from the test statistics. In the 

presence of S9, there was no clear repeat of the toxicity observed with the test material in 

Experiment 1, and only reduction of Day 0 and Day 2 viabilities and relative total growth values 

were observed. 

DETDA induced a statistically significant concentration-related increase in the mutant frequency 

per viable cell in the presence of metabolic activation. In the absence of metabolic activation, an 

increase was observed but at concentration levels with unacceptable toxicity. As with 

Experiment 1, the increase in mutant frequencies was partly due to small colonies, suggesting 

clastogenic activity.

Experiment 3: A 3-hour pulse exposure with S9 was carried out to confirm the positive response 

observed in Experiments 1 and 2. There was evidence of a concentration-related reduction in 

Day 0 relative survival, Day 0 and Day 2 viabilities, and relative total growth.

DETDA induces a statistically significant concentration-related increase in mutant frequency per 

viable cell, although the maximum concentration tested, 1300 μg/mL, was excluded from the 

data due to excessive toxicity. The increase in mutant frequency was partly due to small colony 

formation suggesting clastogenic activity. 

CONCLUSIONS

The negative control and positive control fulfilled the requirements for determination of a valid 

test.

Under the conditions of the assay described in this report, the data suggest that DETDA induces

mutations at the thymidine kinase locus in the presence of metabolic activation and should be 

considered positive in the mouse lymphoma gene mutation assay.

STUDY RELIABILITY

[2] Reliable with restrictions. The report was supplied as an IUCLID summary and important 

details were missing. The summary defines the medium R10 but does not define medium R0 or 
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medium R20. As appropriate under 40 CFR 799.9530 (TSCA in vitro mammalian cell gene 

mutation test) guidance, the study summary included the numbers of small and large mutant 

colonies; however, the IUCLID summary did not indicate whether the original study presented 

mutation frequency by colony size. 

REFERENCES

Cole, J., Fox, M., Garner, C, McGregor, D.B., and Thacker, J. 1990. “Gene mutation assays in 

cultured mammalian cells.” IN: Basic Mutagenicity Tests UKEMS Recommended Procedures 

(Ed. Kirkland, D.J.), Cambridge University Press, pp 87-114.

Durward, R. and Nolan, S. 2000. LZ1224.20: L5178Y TK +/- Mouse Lymphoma Assay, SPL 

Project Number: 102/301. Study No. 3170. Performing Laboratory: SafePharm Laboratories, 

Ltd. Shardlow, Derbyshire. Sponsor: Lonza Limited, Basle, Switzerland. (Cited as Durwood 

and Nolan [2000] in summary submitted by Albemarle and Lonza on March 1, 2012)
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Study 13: Mutagenicity studies in Salmonella typhimurium (Naismith and Matthews, 1979)

Title: Ames Salmonella/microsome plate test

This study is: Adequate Not Adequate

This study is not adequate because the test substance composition is not adequately defined; it is 

referred to only as “DETA”. Other missing details include purity of the test substance and 

statistical methods used.

TEST SUBSTANCE 

Identity: Test substance is referred to only as “DETA”; Purity not specified

Remarks: Dark brown liquid. 

METHOD

Method/guideline followed: PH-301/340

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1979

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 8, 40, 200, 1000 and 5000 μg/plate (preliminary toxicity screen); 0 

(DMSO solvent control), 10, 33, 100, 333 and 1000 μg/plate (mutation assay)

Statistical methods used: Not indicated

Test method/conditions remarks: DETA was miscible in DMSO. It was also noted that

extended contact with 70% ethanol and distilled water caused both solutions to turn blue after 

several hours. Dose levels for the Preliminary Toxicity Screen were 5000, 1000, 200, 40 and 8 

μg/plate. Strain TA1538 showed complete inhibition at 5000 μg/plate and abnormal background 

lawn growth at all other levels. Strain TA100 showed complete inhibition at 5000 μg/plate and 

moderate inhibition at 1000 and 200 μg/plate. Dose levels for DETA in the plate assay were 

1000, 333, 100, 33, and 10 μg/plate. There was 0.07 ml of S-9 supernatant per 1.0 ml of S-9 mix 

used in the rat liver microsomal activation system.
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Tester strains TA1535, TA1537, TA1538, TA98, and TA100 are plated untreated, both with and 

without metabolic activation, to obtain background lawn and revertant colony formation to serve 

as negative controls. Positive controls are strain specific and are as follows: 

TA1535 - without activation - N-Methyl-NPR-Nitro-N-Nitrosoguanidine (20 μg/ml)

TA1537 - without activation - 9-aminoacridine (1500 μg/ml)

TA1538 - with activation - 2-aminofluorene (100 μg/ml)

TA98 - with activation - 2-aminofluorene (100 μg/ml)

TA100 - without activation - N-Methyl-NPR-Nitro-N-Nitrosoguanidine

Tester strains TA1535, TA1537, TA1538, TA98, and TA100 are treated with five levels of the 

test compound. All negative and positive controls are plated in triplicate. All treated groups are 

plated in duplicate.

RESULTS

The results for DETA were negative in strains TA1535, TA1537, TA1538, TA98, and TA100 of 

Salmonella typhimurium in the Ames Test both with and without metabolic activation at 1000, 

333, 100, 33, and 10 μg/plate. 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 8 μg/plate

Results remarks: All negative and positive controls were within one standard deviation of 

historical data.

CONCLUSIONS

The test substance was not mutagenic in S. typhimurium strains TA1535, TA1537, TA1538, 

TA98 or TA100 with or without metabolic activation.

STUDY RELIABILITY

[3] Not reliable. The test substance composition is not adequately defined; it is referred to only 

as “DETA”. Other missing details include purity of the test substance and statistical methods 

used. 

REFERENCES

Naismith, R., and Matthews, R. 1979. Summary Data from Ames Salmonella/Microsome Plate 

Test With and Without Metabolic Activation on DETA. Performing Laboratory: Pharmakon 

Laboratories, Scranton, PA. Sponsor: Ethyl Corporation.
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Study 14: Mutagenicity study in Salmonella typhimurium (Lawlor, 1985)

Title: Salmonella/mammalian-microsome plate incorporation mutagenicity assay

This study is: Adequate Not Adequate

This study is not adequate for the following reasons: (1) Important details are missing from the 

study including purity and composition of the test substance, method/guideline followed, test 

concentrations used in the mutagenicity assay, statistical methods used, analytical data to verify 

actual concentrations used in the assay, cytotoxicity and the results section for the mutagenicity 

assay. (2) According to guideline recommendations, if 2-aminoanthracene is used as a positive 

control, each batch of S9 should also be characterized with a mutagen that requires metabolic 

activation by microsomal enzymes; however, this assay employed 2-aminoanthracene as the sole 

indicator of the efficacy of the S9-mix. (3) Results of positive control assays were not provided.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Referred to as 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Also referred to as MA #T4018; Clear, dark red syrupy liquid

METHOD

Method/guideline followed: Not indicated

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1985

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 0 (DMSO solvent control), 10, 33, 67, 100, 333, 667, 1000, 3333, 6667 

and 1000 μg/plate (preliminary toxicity assay); test concentrations for the mutagenicity assay 

were not specified

Statistical methods used: None specified

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

based on those described by Ames, B, N., et al. Methods for detecting carcinogens and mutagens 

with the Salmonella/Mammalian- Microsome Mutagenicity Test, Mutation Research 31: 347-

364, 1975.
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Media Preparation

Top Agar for Selection of Histidine Revertants: Minimal top agar was prepared with 8 g/liter 

Difco Bacto Agar and 5 g/liter NaCl. After autoclaving, the molten agar was distributed into

sterile bottles and stored at room temperature. Immediately before its use in the mutagenicity 

assay, the top agar was melted and supplemented with 10 ml/100 ml agar of a sterile solution

which contained 0.5 mM L-histidine and 0.5 mM D-biotin. Twenty-five ml of sterile deionized 

water was added per 100 ml top agar when it was used in assays without metabolic activation. 

This ensured that final top agar and amino acid supplement concentrations were the same on 

plates with or without metabolic activation.

Top Agar for Viable Count Determination: Minimal top agar as described above was 

supplemented with 35 m1/100 ml agar of a sterile solution which contained 1.4 mM L-hisitidine 

and 0.12 mM D-biotin.

Minimal Bottom Agar: Bottom agar was Vogel-Bonner minimal medium E.

Nutrient Broth: Nutrient broth used for growing overnight cultures of the tester strains was 

Vogel-Bonner salt solution supplemented with 25g per liter of Nutrient Broth No. 2 (Oxoid).

Nutrient Bottom Agar: Nutrient bottom agar was Vogel-Bonner minimal medium E 

supplemented with 25 g per liter of Nutrient Broth No. 2 (Oxoid).

Tester Strain Diluent: Diluent for tester strain titering contained Vogel-Bonner salt solution with 

25 g per liter of Nutrient Broth No. 2 (Oxoid).

Test Article Diluent: The solvent used for diluting the test article was dimethylsulfoxide (CAS 

#67-68-51, Fisher Scientific Company, Certified A.C.S.

Tester Strains: The tester strains used were histidine auxotrophs TA98, TA100, TA1535, 

TA1537 and TA1538 as described by Ames. 

The test article was checked for toxicity to the tester strains up to a concentration of 10 mg/plate. 

An aliquot from ten dilutions of the test article was plated with an overnight TA100 culture on 

selective minimal agar, both in the presence and absence of metabolic activation. Toxicity is 

detectable by a decrease in the number of revertant colonies occurring per plate and/or by a 

thinning or disappearance of the background bacterial lawn. The highest concentration of test 

article used in the subsequent mutagenicity assay was that which gave a detectable reduction in 

the number of revertants per plate and/or produced a thinning or disappearance of the 

background bacterial lawn.

When necessary, separate dose levels are plated for the portion of the assay with metabolic 

activation and the portion of the assay without metabolic activation.

Plating Procedures for the Mutagenicity Assay Testing System Identification: Each plate was 

labeled using indelible ink with a code system which identifies the test article, test phase, dose 
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level and activation as described in detail in Microbiological Associates, Inc.'s Microbial

Mutagenesis Standard Operating Procedures.

Test Article: The test article was solubilized and serially diluted immediately before its use in the 

mutagenicity assay. Five doses of the test article were plated with all five testes strains (TA98, 

TA100, TA1535, TAl537 and TA1538) with metabolic activation and without metabolic 

activation. All positive controls, solvent controls and test article doses were plated in triplicate.

Without metabolic activation, 50 μl of tester strain and 50 μl of solvent or test article were added 

to 2.5 ml of molten selective top agar at 45°C. With metabolic activation, 50 μl of tester strain, 

50 μl of solvent or test article, and 0.5 ml of S-9 mix were added to 2.0 ml of molten selective 

top agar at 45°C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal 

bottom agar. After the overlay had solidified, the plates were inverted and incubated for 48 hours

at 37°C. Plates which were not counted immediately following the 48-hour incubation period 

were stored at 4°C until such time that colony counting could be conducted.

Positive Controls: All combinations of positive controls and tester strains plated along with the 

assay are listed below:

Strain Activation Positive Controls Conc. per Plate

TA98 + 2-Aminoanthracene 4.0 μg

TA98 - 2-Nitrofluorene 5.0 μg

TA100 + 2-Aminoanthracene 4.0 μg

TA100 - Sodium Azide 5.0 μg

TA1535 + 2-Aminoanthracene 4.0 μg

TA1535 - Sodium Azide 5.0 μg

TA1537 + 2-Aminoanthracene 4.0 μg

TA1537 - 9-Aminoacridine 75 μg

TA1538 + 2-Aminoanthracene 4.0 μg

TA1538 - 2-Nitrofluorene 5.0 μg

9-Aminoacridine (CAS #134-50-9), Aldrich Chemical Co., 98% pure

2-Aminoanthracene (CAS #613-13-8), Sigma Chemical Co., practical grade

2-Nitrofluorene (CAS #607-57-8), Aldrich Chemical Co., 98% pure

Sodium Azide (CAS #26628-22-8), Sigma Chemical Co., practical grade

Tester Strain Titers: Tester strain titers were determined by viable count assays on nutrient agar 

plates. The averaged number of cells plated per plate are reported on the individual strain data 

forms.

Test Article Sterility Determination: The most concentrated test article dilution for the 

mutagenicity assay was checked for sterility by plating a 50 μl aliquot of the dilution on selective 

agar.

Preparation of S-9 Homogenates: Liver microsomal enzymes were prepared from male SpragueDawley rats that had been injected with Aroclor 1254 at 500 mg/kg. The Aroclor was diluted in 
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corn oil to a concentration of 200 mg/ml. Five days after their i.p. injection with the Aroclor, the 

rats were sacrificed by decapitation, and their livers were excised.

The preparation of the microsomal enzyme fraction was carried out with sterile glassware and 

solutions at 0-4°C. The liver from each rat was excised and placed in approximately 20 ml of 

0.15M KCl contained in a pre-weighed beaker. After weighing the liver, it was transferred to 

another beaker containing 3 volumes of 0.15M KCl (3 ml/g of wet lives) where it was minced 

with sterile scissors. The minced liver was homogenized and centrifuged at 9000 x g for 10 

minutes. The supernatant (referred to by Ames as the S-9 fraction) was decanted, and small 

aliquots were distributed into freezing ampules which were stored at ≤ -70°C.

Preparation of S-9 Mix: The S-9 mix was prepared immediately before its use in the 

mutagenicity assay.

Colony Counting: Revertant colonies for a given tester strain within a given test article dilution 

series were counted either entirely by automated colony counter or entirely by hand. Plates with

sufficient test article precipitate to interfere with automated colony counting were counted 

manually.

The condition of the background bacterial lawn was evaluated for evidence of test article 

toxicity, by using a dissecting microscope. This toxicity was scored relative to the solvent control 

plate and recorded along with the revertant count for that plate on the individual strain data 

forms.

Analysis of Data: All platings were done in triplicate. For each triplicate plating, an average and 

standard deviation were calculated. The calculations were done on a Hewlett-Packard HP 25 

programmable calculator.

Evaluation of Mutagenicity Assay Data: For a test article to be considered positive, it must cause

at least a doubling in the mean revertants per plate of at least one tester strain. This increase in 

the mean number of revertants per plate must be accompanied by a dose response to increasing 

concentrations of the test article. In those cases where the-observed dose-responsive increases in 

TA1537 or TA1538 revertants per plate is less than three-fold, the response must be 

reproducible.

Stability of the Test Article: The stability of the test article under the actual experimental 

conditions used in this study was not determined by Microbiological Associates, Inc.

RESULTS

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Not indicated

Results remarks: None specified
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CONCLUSIONS

Diethyltoluenediamine (MA #T4018) did cause positive dose responsive increases with tester 

strains TA98, TA100 and TA1538, only in the presence of Aroclor-induced rat liver microsomes.

STUDY RELIABILITY

[3] Not reliable. Important details are missing from the study including purity and composition 

of the test substance, method/guideline followed, test concentrations used in the mutagenicity 

assay, statistical methods used, analytical data to verify actual concentrations used in the assay,

cytotoxicity and the results section for the mutagenicity assay. According to guideline 

recommendations, if 2-aminoanthracene is used as a positive control, each batch of S9 should 

also be characterized with a mutagen that requires metabolic activation by microsomal enzymes; 

however, this assay employed 2-aminoanthracene as the sole indicator of the efficacy of the S9-

mix. Furthermore, results of positive control assays were not provided.

REFERENCES

Lawlor, T. 1985. Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay. 

Study No. T4018.501015. Performing Laboratory: Microbiological Associates, Inc., Bethesda, 

MD. Sponsor: American Cyanamid Company, Wayne, NJ.
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Study 15: Mutagenicity studies in Salmonella typhimurium (Schechtman et al., 1980)

Title: Activity of T1566 in the Salmonella/microsomal assay for bacterial mutagenicity 

This study is: Adequate Not Adequate

This study is considered to be unreliable because only 2 bacterial strains were tested and because 

the test substance is identified as “diethyl toluene amine”, which is not the chemical specified in 

the test rule 

TEST SUBSTANCE 

Identity: 94% DETA (“diethyl toluene amine”)

Remarks: Also referred to as T1566; Clear dark red liquid; Lot OB#999-79; Known impurities 

include monoethyl toluene diamines. 

METHOD

Method/guideline followed: Not indicated

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Not indicated

Year study performed: 1979

Test species: Salmonella typhimurium strains TA1535 and TA100 

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 0 (negative bacteria only control and DMSO solvent control), 1, 3, 5, 7 

and 10 μl/plate 

Statistical methods used: The numbers of revertant colonies from each set of triplicates were 

averaged and the standard deviation calculated. The average number of revertant colonies found 

in the test compound plates was compared to those found with the solvent control. A compound 

is considered a mutagen if a dose related increase in the number of revertant colonies is found in 

the treatment groups. The first dose level considered for the increase must have an average 

number of revertant colonies which is three times that of the solvent control.

Test method/conditions remarks: The purpose of this study was to employ the Salmonella/

microsomal assay to investigate the mutagenic potential of the test article that has been 

designated in this final report as T1566, otherwise known as DETA.
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A. Mutagenesis Assay Without Metabolic Activation: The assay was performed by the method 

of Ames et al. with the addition of several recommendations. The five bacterial tester strains 

were identified and characterized for the genetic markers described by Ames et al. Briefly, broth 

cultures of S. typhimurium tester strains were grown at 37 ± 1°C for 16-20 hours and 100 μl 

containing 1-3 x 108 bacteria were added to 2 ml molten top agar supplemented with 0.6% NaCl 

and 0.05 mM histidine·HCl and 0.05 mM biotin. The appropriate dose of test chemical, solvent, 

or positive control (100 μl) was added. The tubes were vortex-mixed and the contents poured 

onto a base plate of Spizizen’s minimal medium. The overlay agar was evenly distributed over 

the base plate and allowed to harden. The test chemical was tested at five dose levels against all 

five strains of bacteria. Controls of bacterial only were included for spontaneous reversion. All 

treatments were performed in triplicate. Plates were incubated 48 hours at 37 ± 1°C after which 

time the number of revertant colonies were counted on a Biotran II automatic colony counter.

Due to the facts that colonies around the periphery of the plates are masked and that overlapping 

colonies are not effectively counted, the machine counts were adjusted t o compensate for the 

resulting tendency of automatic counts to be somewhat lower than manual counts. The 

following formula: Y = -7.4 + 1.606X – (7.1 x 10-4

) X2 + (6.31 x 10-7) X3, where Y = corrected 

counts and X = machine counts, was found to transform the machine counts to values which 

accurately reflected manual counts , It was found that no correction was necessary for machine 

counts less than 12. All machine counts greater than 1935, i.e. adjusted counts greater than 5000 

were reported as too numerous to count (TNTC).

B. Mutagenesis Assay With Exogenous Metabolic Activation: The S-9 was prepared according 

to established procedures. Adult male Sprague-Dawley rats, weighing 200-300 gm, were 

induced by a single intraperitoneal injection of Aroclor-1254 at a dosage of 500 mg/kg body 

weight five days before sacrifice. The animals were sacrificed and their livers aseptically 

removed. The excised tissue was rinsed three times in cold sterile 0.15 M KCl and then 

homogenized in a Polytron Tissuemizer at a concentration of 1:3 w/v in 0.15 M

KCl. The supernatant fraction (i.e., S-9) was collected, portioned into aliquots for daily use, and 

stored frozen at -70°C until use. Each bulk preparation of S-9 is assayed for aryl hydrocarbon 

hydroxylase activity and total protein content prior to use.

On the day of the assay, the S-9 was mixed with the co-factor pool to contain: S-9 (100 μl/ml 

cofactor pool), 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP and 0.1 M 

sodium phosphate, pH 7.4. S-9 mix was sterile filtered and stored on ice until used.

The mutation assay was performed in the same way as that described in Section A, except 0.5 ml 

of the S-9 mix was added to the overlay agar immediately before it was mixed and poured. The 

test chemical was tested at five dose levels against all five strains of bacteria. Controls of 

bacteria only were included for spontaneous reversion. All treatments were performed in 

triplicate. All five concentrations of the test chemical and the S-9 mix were checked for sterility. 

Plates were incubated 48 hours at 37 ± 1°C after which time the number of revertant colonies 

were counted on a Biotran II automatic colony counter, All machine counts between 12 and 1935 

were corrected by the following formula: Y = -7.4 + 1.606X - (7.1 x 10-4

) X2 + (6.31 x 10-7) X3, 

where Y = corrected counts and X = machine counts. No correction was required for machine 
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counts less than 12 and all machine counts greater than 1935 were reported as too numerous to 

count (TNTC). 

Controls: In the assay without exogenous metabolic activation, sodium azide (30 μg/plate) was 

used as the positive control for TA1535 and TA100, 9-aminoacridine (100 μg/plate) for TA1537, 

and 2-nitrofluorene (5 μg/plate) for TA1538 and TA98. For the assay with exogenous metabolic 

activation, 2-aminoanthracene (2.5 μg/plate) was used as the positive control for all five strains. 

The solvent vehicle for the test agent was used full strength (100 μl/plate) as the negative

control.

Criteria for Determination of a Valid Test: All sterility controls, including the five concentrations

of the test compound and S-9 mix, must be negative for bacterial growth,

The average number of revertant colonies representing spontaneous mutations must be within the 

following acceptable range: TA1535, 6-60; TA1537, 3-16; TA1538, 6-35; TA100, 80-250;

TA98, 10-60.

The average number of revertant colonies in the positive control plates (i.e. sodium azide for 

TA1535 and TA100, 9-aminoacridine for TA1537, and 2-nitrofluorene for TA1538 and TA98 

for the assay without exogenous metabolic activation; 2-aminoanthracene for all strains in the 

presence of exogenous metabolic activation) must be at least five times those of the solvent 

control.

RESULTS

The positive control for each of the five strains tested induced an average number of 

revertants/plate which was at least five times that found with the solvent control. All 

concentrations of T1566 tested failed to induce an average number of revertants/plate three times 

greater than that found in the solvent control. 

The sterility controls for the S-9 mix, positive controls, and the test article dilutions were all 

negative for bacterial contamination.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): > 10 μl/plate based on no cytotoxicity observed at the 

highest concentration

Results remarks: The negative controls, positive controls, and sterility controls all fulfilled 

requirements for determination of a valid test.

CONCLUSIONS

Under the conditions employed in the assay described above, the data suggest that the test article 

exhibits little or no mutagenic activity in the Salmonella/microsomal assay.
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STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 2 bacterial strains were 

tested and because the test substance is identified as “diethyl toluene amine”, which is not the 

chemical specified in the test rule. In addition, important study details were missing from the 

study, including purity and composition of the test substance, GLP compliance and test 

method/guidelines followed.

REFERENCES

Schechtman, L., Curren, R., and Pamar, A. 1980. Activity of T1566 in the Salmonella/

Microsomal Assay for Bacterial Mutagenicity Final Report. Performing Laboratory: 

Microbiological Associates. Bethesda, MD. Sponsor: Ethyl Corporation.
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Study 16: Mutagenicity studies in Salmonella typhimurium (Johnson and Pettigrew, 1987b)

Title: Salmonella/microsomal assay with darkened DETDA - low color (Lot #6198-20-1)

This study is: Adequate Not Adequate

This study is considered to be unreliable because only 4 bacterial strains were tested. 

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Referred to as darkened DETDA - low color, Lot #6198-20-1; Clear brown liquid. 

METHOD

Method/guideline followed: Protocol - Ettox 027

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

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

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 0 (untreated control), 0 (DMSO solvent control), 10, 37.5, 75, 100 and 

125 mg/mL

Statistical methods used: Not indicated

Test method/conditions remarks: Salmonella typhimurium strains TA1535, TA1537, TA98 

and TA100 were treated in the presence of metabolic activation (Aroclor 1254 induced rat liver 

S-9 fraction) with Darkened DETDA-Low Color in DMSO. Dose levels tested were 125, 100, 7 

5, 37.5, and 10 mg/mL.

Darkened DETDA-Low Color was soluble at all dose levels tested. Each dose was treated in 

triplicate. An untreated control, solvent control and positive control were treated concurrently.

RESULTS

125 mg/mL demonstrated toxicity in strain TA1535 with metabolic activation. This toxicity 

caused the apparent increase in revertant colonies in strain TA1535. Darkened DETDA-low 
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color did not cause a dose related increase in mutant colonies in any other strain in the presence 

of metabolic activation.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 125 mg/mL in strain TA1535 with metabolic 

activation

Results remarks: Color and appearance of the test article did not change from the time of 

receipt to the time of use.

CONCLUSIONS

Darkened DETDA-Low Color was not genetically active in the Salmonella/ Microsomal Assay 

according to Protocol - Ettox 027.

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 4 bacterial strains were 

tested. In addition, important study details were missing from the study summary provided,

including purity and composition of the test substance and statistical methods used.

REFERENCES

Johnson, D., and Pettigrew, A. 1987b. Genetic toxicology. Salmonella/Microsomal Assay with 

Darkened DETDA - Low Color (Lot #6198-20-1). Ames 101 - #112. Sponsor: Ethyl 

Corporation, Baton Rouge, LA.
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Study 17: Mutagenicity studies in Salmonella typhimurium (Johnson and Pettigrew, 1987c)

Title: Salmonella/microsomal assay with DETDA-low color

This study is: Adequate Not Adequate

This study is considered to be unreliable because only 4 bacterial strains were tested. 

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediame (DETDA); Purity not specified

Remarks: DETDA-Low Color, Lot # 5869-75-1 and DETDA-Obg, Lot # 5868-1; Clear liquid. 

METHOD

Method/guideline followed: Protocol Ettox- 027

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

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

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 0 (DMSO solvent control), 750, 1000, 3750, 7500 and 10,000 μg/plate

Statistical methods used: Not indicated

Test method/conditions remarks: Salmonella typhimurium strains TA1535, TA1537, TA98 

and TA100 were treated in the presence of metabolic activation (Aroclor 1254 induced rat liver 

S-9 fraction) with DETDA-Low Color in DMSO. Dose levels tested were 10,000, 7500, 3750, 

1000, and 750 μg/plate. DETDA-Low Color was soluble at all dose levels tested. Each dose

was treated in triplicate. An untreated control, solvent control and positive control were treated 

concurrently.

DETDA-Obg (Lot #5868-1) was tested in the same strain s and at the same dose levels as 

DETDA-Low Color.
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RESULTS

DETDA-Low Color did not cause a dose related increase in mutant colonies in any strain in the 

presence of metabolic activation. DETDA-Obg caused a dose related increase in mutant 

colonies in TA1535, TA1537, TA98, and TA100 in the presence of metabolic activation.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not indicated

Results remarks: Color and appearance of the test article did not change from the time of 

receipt to the time of use.

CONCLUSIONS

DETDA-Low Color was not genetically active in the Salmonella/Microsomal Assay according to 

Protocol – Ettox 027.

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 4 bacterial strains were 

tested. In addition, important study details were missing from the study summary provided and 

included purity and composition of the test substance, statistical methods used and cytotoxicity 

of test substance.

REFERENCES

Johnson, D., and Pettigrew, A. 1987c. Genetic Toxicology. Salmonella/Microsomal Assay with 

DETDA-Low Color. Ames 099 - #111. Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 18: Mutagenicity studies in Salmonella typhimurium (Stankowski and Naismith, 

1987b)

Title: Ames/Salmonella plate incorporation assay with DETDA-low color Lot # 2-87

This study is: Adequate Not Adequate

This study is considered to be unreliable because only 3 bacterial strains were tested. 

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Lot # 2-87; Clear liquid. 

METHOD

Method/guideline followed: Protocol- 301

Test type: Ames Salmonella reverse gene mutation assay

GLP compliant?: Yes

Year study performed: 1987

Test species: Salmonella typhimurium strains TA1538, TA98 and TA100 

Metabolic activation: With S-9 metabolic activation from rat livers

Concentrations tested: 

Assay 1: 500, 750, 1000, 3750 and 7500 μg/plate 

Assay 2: 1000, 3750, 7500, 10,000 and 12,500 μg/plate

Statistical methods used: A positive result is defined as a reproducible, statistically significant 

two-fold dose-dependent increase in the number of histidine-independent colonies, with at least 

one dose point inducing an average revertant frequency that is two-fold that of the solvent 

control. Significance is determined using the program developed by Moore and Felton (1983). 

This program applies a linear regression analysis to the data points and any p value less than 0.05 

is considered significant. Alternatively, if the test article produces an increase greater than or 

equal to three times the solvent control value (in the absence of a dose-dependent increase), the 

test chemical is considered positive. A negative result is defined as the absence of a dosedependent two-fold increase in the number of histidine-independent revertants.

Test method/conditions remarks: DETDA-Low Color was evaluated in triplicate cultures in 

strains TA1538, TA98 and TA100 in the presence of S9 at doses of 500, 750, 1000, 3750 and 
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7500 μg/plate. The S9 mixture included 6% (v/v) Aroclor 1254-induced male Sprague-Dawley 

rat liver homogenate with the appropriate buffer and cofactors. Revertant frequencies for all

doses of DETDA-Low Color in all strains approximated or were less than those observed in the 

concurrent negative control cultures. However, no toxicity was observed at the highest dose level

evaluated. Therefore, DETDA-Low Color was re-evaluated in all three strains at doses of 1000, 

3750, 7500, 10,000 and 12,500 μg/plate with S9. Also evaluated in this second assay was a 

concurrent positive control was a second sample of DETDA (Drum #6131). This latter sample of 

DETDA was previously found to be mutagenic in the Ames/Salmonella Plate Incorporation 

Assay.

Treatment with Test and Control Articles: Treatments were performed by combining 2 ml top 

agar (supplemented with 0.5mM histidine/0.5mM biotin), 0.1 ml tester strain, 0.5 ml of the S9 

mixture and 0.1 ml test or control article or solvent in sterile glass tubes preheated to 45°C. The 

S9 mixture contained 8mM MgCl2, 33mM KC1, 4mM NADP, 5mM glucose-6-phosphate, 

100mM NaH2 PO4 (pH 7.4) and 6% (v/v) Aroclor 1254-induced male Sprague-Dawley rat liver 

homogenate. The tubes were vortexed end the mixture was poured onto minimal glucose plates, 

evenly distributed, and allowed to solidify. Within an hour the plates were inverted and 

incubated in the dark at 37°C for 48-72 hours.

Bacterial Contaminant Evaluation: To ensure the sterility of solvents, compounds and 

equipment, standard contamination evaluations were performed with the assay. The solvent, top

agar, S9 mix, and top dose of the test article were evaluated at the same volumes used in the 

assay. The test article, solvent and S9 mix were evaluated as in the mutation assay, but in the 

absence of added Salmonella. Top agar alone was also plated on minimal glucose plates. All

plating was done in triplicate. Plates were incubated for 48-72 hours at 37°C and then scored for 

bacterial growth.

Scoring: Following the 48-hour incubation, the background lawn and spontaneous revertants 

were scored for normal, inhibited or no growth. Inhibited growth was characterized by the 

absence of a confluent bacterial lawn and/or the presence of pindot colonies. Revertant colonies 

were enumerated on an Artek electronic colony counter interfaced with an Apple computer for 

data acquisition. Solvent and positive controls were scored first, and test article treated cultures 

were scored only if the average negative control values were within historical ranges.

RESULTS

Revertant frequencies for all doses of DETDA-Low Color in all strains in the second assay 

approximated or were less than those observed in the concurrent negative control cultures. In 

addition, all strains exhibited reduced revertant frequencies and background lawns at the 12,500 

μg/plate dose level, indicating this dose was moderately toxic. 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 12500 μg/plate

Results remarks: All positive and negative control values in both assays were within historical 

limits.
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CONCLUSIONS

The results for DETDA-Low Color were negative in the Ames/Salmonella Plate Incorporation 

Assay under the conditions, and according to the criteria, of the test protocol.

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 3 bacterial strains were 

tested. In addition, important study details were missing from the study summary provided and 

included purity and composition of the test substance.

REFERENCES

Stankowski, L., and Naismith, R. 1987b. Ames/Salmonella Plate Incorporation Assay with 

DETDA-Low Color Lot # 2-87. Report # PH 301-ET-001-87. Performing Laboratory: 

Pharmakon Research International, Waverly, PA.
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Study 19: In vitro point mutation in mammalian cells (Schechtman et al., 1982)

Title: Activity of diethyl toluenediamine (DETDA) in the in vitro mammalian cell point 

mutation assay in the absence of exogenous metabolic activation

This study is: Adequate Not Adequate

This study is not adequate because it did not indicate a test guideline, did not indicate the purity 

and composition of the test substance and did not use at least four analyzable concentrations of 

test substance that produced a range of toxicity from maximum to little or no toxicity. 

Additionally, the cells that were tested (BALB/3T3 clone A31-1 mouse cells) are not named in 

OPPTS 870.5300 as one of the cell lines suitable for detection of gene mutations in mammalian 

cells and the test substance was not tested in the presence of metabolic activation.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA) 

Remarks: DETDA lot PP Run 162 assigned the code number T1829; purity not provided; 

viscous brown liquid in appearance

METHOD

Method/guideline followed: Not indicated

Test type: In vitro mammalian cell point mutation

GLP compliant?: Yes

Year study performed: 1982

Test species: BALB/3TC Clone A31-1 mouse cells

Metabolic activation: Without

Concentrations tested: 0.05, 0.17, or 0.5 μl/ml 

Statistical methods used: The mutagenic potential of each treatment condition was compared 

to that of the solvent control using a special application of the Poisson distribution as follows:

The proportion (P) of all cells not subject to induced mutation is equal to the number of clonable 

cells in the solvent control group divided by the total of the clonable cells in the solvent control 

and treatment group. Significance is determined by:

P(X≥n1/n) = (n!/(n1!(n-n1)!)) (Pn1 (1-P)n-n1)
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Where p is the probability, n is the number of induced mutants, n1 is the number of spontaneous 

mutants, and P is defined above. If p is very small (p ≤ 0.05) the hypothesis that the probability 

of a mutation event is the same for both the treated and control groups is rejected and the induced 

mutation frequency is considered significant at the p ≤ 0.05 level.

Test method/conditions remarks: 

Exponentially grown BALB/3T3 clone A31-1 cells were seeded for each treatment condition at 

approximately 2 x 106 cells/75 cm2 flask and were incubated at 36oC in a humidified atmosphere 

of 5% CO2 in air for 20-24 hours.

The time of initiation of chemical treatment was designated as day 0. The test substance was 

dissolved in DMSO and then diluted with complete EMEM (Eagles Minimal Essential medium 

supplemented with 10% fetal bovine serum, 1% L-glutamine and 2% penicillin-streptomycin). 

Cells were exposed to three concentrations of the test article as well as solvent and positive 

controls for 24 hours at 36oC. Treatment was carried out by adding 2X concentrations of test 

article, solvent, or positive control to an equal volume of complete EMEM in the flask. After the 

exposure period, all treatment media were withdrawn, the cells were washed once with HBSS, 

collected by trypsinization, and subcultured to assess cytotoxicity and to initiate the phenotypic 

expression period. 

For evaluation of cytotoxicity, cells from each treatment condition were subcultured in complete 

EMEM, in triplicate, at a density of 250 cells/dish. After 7-10 days incubation at 36oC, the 

colonies were fixed with methanol, stained with 10% aqueous Giemsa, and counted.

For expression of the mutant phenotype, cells from each treatment condition were subcultured in 

complete EMEM at a density of approximately 4 x 105 cells/150 cm2 flask. After a 4-6 day 

expression period, selection for the mutant phenotype was performed.

For selection of the ouabain-resistant phenotype, cells from each treatment conditions were 

replated, in replicates of 15 dishes, at a density of 2 x 105 cells/60 mm dish in complete EMEM 

containing 1 mM ouabain. After 1-4 weeks, the mutant colonies were fixed, stained, and 

counted. For cloning efficiency at the time of selection, 250 cells/60 mm dish were plated, in 

triplicate, in selection medium without ouabain. After 7-10 days incubation at 36oC, the colonies 

were fixed, stained and counted for cloning efficiency.

Evaluation of Test Results – The cytotoxic effects of each treatment condition are expressed 

relative to the solvent-treated control (relative cloning efficiency). The mutation frequency for 

each treatment condition is expressed as the number of mutant colonies per clonable cell. The 

confidence level associated with the number of clonable cells employed in the selection process 

requires that the assumption be made that were more cells utilized for the selection of mutants, 

the possibility exists that at least one ouabain-resistant mutant could be obtained. Therefore, for 

test conditions in which no mutant colonies were observed, mutation frequencies are expressed 

as less than the frequency obtained with one mutant colony.

Criteria for Determination of a Valid Test – The cloning efficiency of the solvent control must be 

greater than or equal to 15%. The relative survival of cells exposed to the test article must be 

equal to or greater than 40% for at least one dose. The number of spontaneous ouabain-resistant 
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mutant colonies in the solvent control must not exceed four colonies per total replicate dishes. 

The positive control must induce a significant (p ≤ 0.05) number of ouabain-resistant mutant 

colonies relative to the negative control.

RESULTS 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Relative to the solvent control, the cell survival was 

approximately 73%, 80%, and 84% at 0.5, 0.17, and 0.05 μl/ml, respectively.

Results remarks: Cytotoxicity was observed. All tests were negative for the induction of 

ouabain-resistant colonies at all concentrations employed.

Following a 24 hour treatment of BALB/3T3 clone A31-1 cells with the test substance in the 

absence of an exogenous metabolic activation system, cell survival (relative to the solvent 

control) was approximately 73%, 80%, and 84% at 0.5, 0.17 and 0.05 μl/ml, respectively.

For the activity of test substance in the BALB/3T3 point mutation system after a 24 hour 

treatment in the absence of an exogenous metabolic activation system, no spontaneous ouabainresistant mutant colonies were observed in the solvent control for a background mutation 

frequency of < 0.79 X 10-6

. No ouabain-resistant mutant colonies were observed in the three 

treatment groups. The mutation frequencies of <0.83 X 10-6

, <0.81 X 10-6 and <0.85 X10-6 were 

not statistically increased (p>0.05) relative to the solvent control. MNNG induced 247 ouabainresistant mutants for a mutation frequency of 217 X 10-6 which was statistically significant 

(p<0.01) to the solvent control.

CONCLUSIONS

The negative control and positive control fulfilled the requirements for determination of a valid 

test.

Under the conditions of the assay described in this report, the data suggest that T1829 does not 

induce point mutations at the ouabain membrane receptor locus and should be considered 

negative in the BALB/3T3 point mutation assay.

STUDY RELIABILITY

[3] Not reliable. The study did not indicate a test guideline, did not indicate the purity and 

composition of the test substance and did not use at least four analyzable concentrations of test 

substance that produced a range of toxicity from maximum to little or no toxicity. Additionally, 

the cells that were tested (BALB/3T3 clone A31-1 mouse cells) are not named in OPPTS 

870.5300 as one of the cell lines suitable for detection of gene mutations in mammalian cells and 

the test substance was not tested in the presence of metabolic activation.
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REFERENCES

Schechtman, L., Putman, D., and McCarvill, J. 1982. Activity of diethyl toluenediamine 

(DETDA) in the in vitro mammalian cell point mutation assay in the absence of exogenous 

metabolic activation. Performing Laboratory: Microbiological Associates. Bethesda, MD. 

Sponsor: Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 2011. 

Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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Study 20: In vitro point mutation in mammalian cells (Schechtman et al., 1979 )

Title: Activity of T1364A in in vitro mammalian cell point mutation assay in the presence of 

exogenous metabolic activation

This study is: Adequate Not Adequate

This study is not adequate because the study did not indicate the test guideline(s) followed, GLP 

compliance and test substance purity and composition. Evidence was not provided, as stipulated 

by the OPPTS 870.5300 guideline for the In Vitro Mammalian Cell Gene Mutation Test, that the 

study used at least four analyzable concentrations that produced a range of toxicity from 

maximum to little or no toxicity or, alternatively, that the highest test concentration was 5 mg/L, 

5μL/mL, or 0.01M, whichever is the lowest. Additionally, the cells that were tested, BALB/3TC 

Cl.A31 mouse cells, are not named in OPPTS 870.5300 as one of the cell lines suitable for 

detection of gene mutations in mammalian cells and the test substance was not tested in the 

absence of metabolic activation. Furthermore, the test substance is identified as 

diethyltolueneamine, which differs from diethyltoluenediamine (the subject of required testing); 

diethyltolueneamine is not an acceptable analog for diethyltoluenediamine.

TEST SUBSTANCE 

Identity: Diethyltolueneamine 

Remarks: Referred to as T1364A; brown liquid in appearance

METHOD

Method/guideline followed: Not indicated

Test type: In vitro mammalian cell point mutation

GLP compliant?: Not indicated

Year study performed: 1979

Test species: BALB/3T3 Cl.A31 mouse cells

Metabolic activation: With (Preliminary cytotoxicity evaluations were conducted with and 

without)

Concentrations tested: 1.1 x 104

, 1:1.5 x 104, 1:2 x 104

Statistical methods used: Not indicated
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Test method/conditions remarks: 

Prior to the use, an S-9 preparation is pre-tested for metabolic capacity in terms of aryl 

hydrocarbon hydroxylase (AHH) activity by medication of published procedures.

Exponentially growing BALB/3T3 Cl.A31 cells were cultured in 75 cm2 cell culture flasks and 

collected by trypsinization into 50 cc conical tubes for treatment in suspension at 37oC for 2 

hours in a buffer pool (reaction mixture) containing an NADPH-generating system. The 

treatment conditions were as follows:

a. Cells + S-9 mix + Benzo(a)pyrene (BP), 12.5 μg/ml (positive control)

b. Cells + S-9 mix ± Dimethylsulfoxide (DMSO) (vehicle/negative control)

c. Cells + S-9 mix + test chemical (3 doses)

After the exposure period, the target cells were washed and resuspended in complete growth 

medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum. Cell 

samples from each test condition were seeded at 250 cells per 60 mm dish in triplicate, to 

determine the cytotoxic effects of each treatment. After 7-10 days at 37oC, the cells were fixed 

with 100% methanol, stained with 10% aqueous Giemsa, and scored for the number of surviving 

colonies in order to determine the relative plating efficiencies (RPE) according to the protocol of 

Schechtman and Kouri (Appendix A). 

The remaining cells for each test condition were cultured further (in 150 cm2 cell culture flasks) 

at 37oC for a 4-6 days expression period necessary for fixation of the mutagenic event. Cell 

samples were then collected and reseeded at 250 cells per 60 mm dish in triplicate, to determine 

their RPE, in order to establish the number of cells at risk (CAR) to the selective agent, ouabain. 

After 7-10 days at 37oC, the cells were fixed, stained and scored as described above.

For determination for mutation induction, cells from each test condition collected postexpression period were also seeded at 2 X 105 cells per 60 mm dish, approximately 15 dishes per 

condition, and incubated at 37oC for 4 hours. One ml of 5mM ouabain was then added to each 

dish, resulted in a final concentration of 1 mM ouabain. Plates were incubated at 37oC with 

scheduled medium changes containing 1 mM ouabain, over a 4-6 week period. Cells were then 

fixed, stained and scored for the development of ouabain-resistant colonies, according to the 

protocol of Schechtman and Kouri (Appendix B).

The results of the mutation assays are expressed in terms of the number of ouabain-resistant 

mutants per number of CAR, or as the total number of ouabain-resistant mutant colonies per 

condition.

A preliminary cytotoxicity study was conducted to determine the dose range to be employed in 

the mutagenesis assay. The detailed protocol for this cytotoxicity assay is included as Appendix 

C. Wherever possible, the approximate LD50 concentration of the test chemical plus 1 dose 

above and 1 dose below the LD50 were those used in the assay. 
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Appendix A

Standard Protocol for the Determination of the Cytotoxic Effects of Test Chemicals to 

BALB/3T3 Cl. A31 Mouse Cells in the Presence of Exogenous Metabolic Activation: 

Suspension Assay

1. Two-3 x 106 logarithmically growing BALB/3T3 Cl. A31 cells (1) from sub-confluent stock 

cultures are collected into 50 cc capacity conical centrifuge tubes (two tubes per treatment 

condition) and centrifuged at 1000 RPM for 10 min at 4 C.

2. The supernatants are discarded and the cell pellets are resuspended in 4 ml reaction mixture 

consisting of the S-9 mix (containing 100 μl S-9 (approximately 40 mg protein/ml) and cofactor 

pool) ± test chemical. The test conditions include: 

a. Cells + S-9 mix + BP, 12.5 (positive control)

b. Cells + S-9 mix ± solvent vehicle (negative control)

c. Cells + S-9 mix + test chemical

Final concentrations of the test chemical employed are those used in the accompanying 

mutation/transformation assay.

3. The tubes containing each reaction mixture are incubated for 2 hr at 37C in a humidified 

atmosphere of 5% CO2 + air. The reaction is terminated by centrifugation at 1000 RPM for 10 

min at 4C.

4. The cell supernatants are discarded; the cell pellets are resuspended for washing in Hanks’ 

balanced salt solution and then centrifuged at 1000 RPM for 10 min at 4C.

5. The cell supernatants are discarded and the cell pellets are resuspended in growth medium 

(basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum. A cell count is then 

taken on the cells pooled for each test condition.

6. Cells are seeded at 250 cells per 60 mm dish in triplicate dishes per condition, in 5 ml growth 

medium and incubated at 37C.

7. The medium in all plates is replaced every 3-4 days for the duration of the assay.

8. The plates are fixed (methanol, 100%) stained (Giemsa, 10% aqueous) and scored 7-10 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded), and/or relative colony-forming efficiency (RCE) (i.e. 

number of colonies in chemically treated dishes relative to the number of colonies in solventtreated controls).

Appendix B

Standard Protocol for the Mutagenesis of BALB/3T3 Cl. A31 Mouse Cells at the OuabainResistance Locus (OuaR

) in the Presence of Exogenous Metabolic Activation: Suspension Assay
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1. Two-3 x106 logarithmically growing BALB/3T3 Cl. A31 cells (1) from sub-confluent stock 

cultures are collected onto 50 cc capacity conical centrifuge tubes (two tubes per treatment 

condition) and centrifuged at 1000 RPM for 10 min at 4 C.

2. The supernatants are discarded and the cell pellets are resuspended in 4 ml reaction mixture 

consisting of the S-9 mix (containing 100 μl S-9 (approximately 40 mg protein/ml) and cofactor 

pool) ± test chemical. The test conditions include:

a. Cells + S-9 mix + BP, 12.5 μg/ml (positive control)

b. Cells + S-9 mix ± solvent control (negative control)

c. Cells + S-9 mix + test chemical (3 doses)

The doses of test chemical used shall be based upon the results obtained in a preliminary 

cytotoxicity assay, and shall include, whenever possible, the approximate LD50, one dose above 

and one dose below the LD50. (The LD50 is that dose which is lethal to 50% of the target cells.) 

If an agent is non-toxic, then the highest dose that will be used will be a function of the limits of 

solubility of the test agent.

3. The tubes containing each reaction mixture are incubated for 2 hr at 37 C in a humidified 

atmosphere of 5% CO2 + air. The reaction is terminated by centrifugation at 1000 RPM for 10 

min at 4 C.

4. The cell pellets are resuspended for washing in Hank’s balanced salt solution and then 

centrifuged at 1000 RPM for 10 min at 4C.

5. The cell supernatants are discarded and the cell pellets are resuspended in growth medium 

(basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum. A cell count is then 

taken on the cells pooled for each test condition.

6. Cytotoxicity assay plates (derived from each test condition) are seeded at 250 cells per 60 mm 

dish, in triplicate dishes per condition in 5 ml growth medium to determine the cytotoxic effects 

of each treatment. Plates are incubated at 37 C. The medium is replaced every 3 days for 7-10 

days, after which the cells are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and 

scored for relative plating efficiency (RPE) (i.e. number of surviving colonies per number of 

cells seeded).

7. The remaining cells from each test condition are seeded into 150 cm2 tissue culture flasks (~5 

x 105 cells/flask) in 25 ml growth medium, and are incubated at 37C for 4-6 days (with medium 

changes every 3 days). This is the period of time required for fixation and expression of the 

mutagenic event.

8. After the expression period, the cells from each test condition are collected by trypsinization 

(using 0.25% trypsin), suspended in growth medium, pooled and counted.

9. Cells are reseeded at 250 cells per 60 mm dish, in triplicate dishes per condition in 5 ml 

growth medium to determine their RPE, in order to establish the number of cells at risk (CAR) to 
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the selective agent (which discriminates mutant cells). These dishes are handled as described in 

step #6 above.

10. For mutagenesis determinations, 2 x 105 cells per 60 mm dish are seeded, approximately 15 

dishes per condition, in 4 ml growth medium. Plates are incubated at 37 C for 4 hr.

11. One ml of 5 mM ouabain is added to each dish to give a final concentration of 1 mM 

ouabain. (Ouabain is a steroid which serves as the selective agent for discriminating mutant 

ouabain-resistant colonies.) Plates are incubated at 37 C for 12 days.

12. The medium in the mutagenesis plates is then replaced once each week with fresh growth 

medium containing 1 mM ouabain for 3-4 weeks.

13. Cells are fixed, stained and scored for the development of mutant ouabain-resistant colonies.

14. Mutation frequency (MF) data can be expressed in one of two ways: either (a) as the number 

of ouabain-resistant mutants per number of cells at risk to the selective agent (which is a function 

of the RPE), or (b) as the total number of ouabain-resistant mutants per condition.

Appendix C

Standard Protocol for the Determination of the Cytotoxic Effects of Test Chemicals to BALB 

3T3 Cl. A31 Mouse Cells in the Absence of Exogenous Metabolic Activation: Plate Assay

1. BALB 3T3 Cl. A31 cells from sub-confluent stock cultures are seeded at 2 different cell 

densities (250 and 500 cells per 60 mm dish) in triplicate dishes per condition, in 4 ml growth 

medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum, and 

incubated at 37 C in a humidified atmosphere of 5% CO2 + air.

2. Twenty-four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle 

(negative control) are added 1:1 (v/v) to the seeding medium; cells are incubated for 24 hr at 37 

C.

3. Final concentrations of test chemical to be tested initially will cover a dose range from 30-0.1 

μg/ml in approximate half-log dilutions. However, if desirable, the highest dose to be tested will 

be determined by the limits of solubility of the test agent.

4. After a 24 hour exposure period, the treatment medium is withdrawn; cells are washed once 

with Hanks’ balanced salt solution (pH 7.0) and resupplied with 5 ml fresh growth medium.

5. The medium in all plates is replaced every 3-4 days for the duration of the assay.

6. The plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and scored 7-10 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded) and relative colony-forming efficiency (RCE) (i.e. number 

of colonies in chemically-treated dishes relative to the number of colonies in solvent-treated 

controls).
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7. The approximate LD50 is determined from the relative survival data. The LD50 is that dose of 

test chemical which is lethal to 50% of the target cells.

RESULTS 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Concentrations ≤ 1:1.15 x 104 were extremely 

cytotoxic in the preliminary cytotoxicity evaluations. Relative survival for the concentrations 

used in the mutagenicity assay (in the presence of metabolic activation) was 43.1%, 89.9% and 

101.9% at 1:1 x 104

, 1:1.5 x 104 and 1:2 x 104, respectively.

Results remarks: Under the conditions of the preliminary cytotoxicity study (Appendix C), 

compound T1364A was extremely cytotoxic to the target cells. The test agent was 100% 

cytotoxic at all of the concentrations tested although a few single cells were observed at the 

1:1000 dilution; however, no colony formation was observed. Compound T1364A precipitated 

heavily at all of the concentrations tested.

In an additional preliminary cytotoxicity assay utilizing revised dilutions, T1364A was observed 

to be 100% cytotoxic at the 1:1 x 103

, 1:2 x 103 and 1:3 x 103 dilutions. Although cell survival 

and colony formation was observed at the 1:5 x 103 dilution, the relative survival of the target 

cells at this concentration was judged to be too low to be used in the mutation assay.

These results necessitated an additional cytotoxicity assay which would serve to estimate the 

approximate LD50 of the test compound in the presence of exogenous metabolic activation. 

Although quantitative results (i.e. colony formation, RPE) were not established, a qualitative 

estimation of the relative toxicity of test agent to the target cells expressed in terms of (%) 

relative survival after treatment could be established. Under these conditions, compound 

T1364A was again observed to be extremely cytotoxic, even at a 1:1 x 104 dilution (relative 

survival ≤ 10%). A dramatic decrease in the level of toxicity was observed at dilutions ≥ 1:1.5 x 

104. The observed cellular response made it difficult to establish an LD50 for test agent T1364a. 

Therefore, the mutagenesis assay (and its accompanying cytotoxicity assay) was performed using 

these same doses in order to bracket the LD50 range.

Under the conditions of the quantitative cytotoxicity study which accompanied the mutagenesis 

assay (Appendix A), an approximate LD60 was observed at the 1:1 x 104 dilution of test 

compound T1364A in the presence of exogenously supplied S-9. Little or no toxic effects were 

observed at the other doses of T1364A employed.

Under the conditions of the mutagenesis assay, no ouabain-resistant colonies were observed at 

any of the doses of T1364 tested. No spontaneous mutant colonies were induced among cells in 

the negative control (S-9 + buffer). In the presence of S-9, BP (the positive control chemical) 

induced 20 ouabain-resistant colonies per 0.58 x 106 cells at risk.
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CONCLUSIONS

While the positive control was seen to induce ouabain-resistant colonies, the test substance did 

not, even at concentrations that were not cytotoxic. Therefore, the test substance was negative 

for mutation in this assay. Additional information is needed to determine if it was negative 

without metabolic activation.

STUDY RELIABILITY

[3] Not reliable. The study did not indicate the test guideline(s) followed, GLP compliance and 

test substance purity and composition. Evidence was not provided, as stipulated by the OPPTS 

870.5300 guideline for the In Vitro Mammalian Cell Gene Mutation Test, that the study used at 

least four analyzable concentrations that produced a range of toxicity from maximum to little or 

no toxicity or, alternatively, that the highest test concentration was 5 mg/L, 5μL/mL, or 0.01M, 

whichever is the lowest. Additionally, the cells that were tested, BALB/3TC Cl.A31 mouse cells, 

are not named in OPPTS 870.5300 as one of the cell lines suitable for detection of gene 

mutations in mammalian cells and the test substance was not tested in the absence of metabolic 

activation. Furthermore, the test substance is identified as diethyltolueneamine, which differs 

from diethyltoluenediamine (the subject of required testing); diethyltolueneamine is not an 

acceptable analog for diethyltoluenediamine.

REFERENCES

Schechtman, L., Beard, S., and Sinsky, P. 1979. Activity of T1364A in in vitro mammalian cell 

point mutation assay in the presence of exogenous metabolic activation. Performing Laboratory: 

Microbiological Associates. Bethesda, MD. Sponsor: Ethyl Corporation, Baton Rouge, LA. As 

cited in Albemarle Corporation. 2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-

0531, MR #335194.
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Study 21: In vitro point mutation in mammalian cells (Schechtman et al., 1978)

Title: Activity of DETA in in vitro mammalian cell point mutation assay

This study is: Adequate Not Adequate

This study is not adequate because the study did not indicate the test guideline(s) followed, GLP 

compliance, and test substance purity and composition. Additionally, the cells that were tested, 

BALB/3TC Cl.A31 mouse cells, are not named in OPPTS 870.5300 as one of the cell lines 

suitable for detection of gene mutations in mammalian cells and the test substance was not tested 

in the presence of metabolic activation.

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediame (DETDA) 

Remarks: Referred to as “DETA”, identified as “diethyltoluenediamine”, compound lot number 

designated as X-9782; brown liquid in appearance

METHOD

Method/guideline followed: Not indicated

Test type: In vitro mammalian cell point mutation

GLP compliant?: Not indicated

Year study performed: 1978

Test species: BALB/3TC Cl. A31 mouse cells

Metabolic activation: Without

Concentrations tested: Not indicated

Statistical methods used: Not indicated

Test method/conditions remarks: Exponentially growing BALB/3T3 Cl. A31 cells were 

cultured in 75 cm2 Falcon flasks and collected by trypsinization. Cells were seeded in triplicate 

dishes per condition at densities of 250 and/or 500 cells per 60 mm dish for determination of the 

relative cytotoxic effects of each treatment and 1 x 104 cells per 60 mm dish in ~ 15 replicate 

dishes per condition for determination of the mutation frequency. All cells were seeded in 4 ml 

complete growth medium.

Twenty-four hours after the cells were seeded, a twice concentrated working solution of the test 

chemical (3 doses) or the positive or negative controls were added 1:1 (v/v) to the medium in 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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each dish; cells were then incubated for 24 hours at 37oC. After the exposure period all 

treatment media were withdrawn and the target cells were washed once with Hanks’ Balance Salt 

Solution (HBSS) supplemented with 5% fetal bovine serum. Thereafter the cells were 

replenished with 5 ml complete growth medium. The medium was changed once during the 

expression period (of 5 days).

Six days after the initiation of the experiment, the dishes which were initially seeded with 1 x 104

cells were treated with medium containing 1 mM ouabain for selection of resistant mutant 

colonies developing over 25-30 days.

Cells were fixed with 100% methanol and stained with 10% aqueous Giemsa after 8-10 days for 

cytotoxicity and plating efficiency (cells at risk) determinations, according to the protocol of 

Schechtman and Kouri (Appendix A), and at 25-30 days for determination of the number of 

mutant colonies in each dish, according to the protocol of Schechtman and Kouri (Appendix B). 

Plating efficiencies are expressed as the percent of the number of surviving colonies per dish 

relative to the number of cells seeded per dish. Mutation results are designated in terms of 

mutation frequencies and are expressed as the number of ouabain-resistant mutants per number 

of cells at risk to ouabain.

A preliminary cytotoxicity study was conducted to determine the dose range to be employed in 

the mutagenesis assay. The detailed protocol for this cytotoxicity assay is included as Appendix 

A. Wherever possible, the approximate LD50 concentration of the test chemical plus 1 dose 

above and 1 dose below the LD50 were those used in the assay. Cell culture (growth) medium, 

acetone, or DMSO, the solvent vehicle, served as the negative control; 3-MC or MNNG was 

used as the positive control.

APPENDIX A

Standard Protocol for the Determination of the Cytotoxic Effects of Test Chemicals to BALB 

3T3 CL. A31 Mouse Cells in the Absence of Exogenous Metabolic Activation: Plate Assay

1. BALB 3T3 Cl. A31 cells from sub-confluent stock cultures are seeded at 2 different cell 

densities (250 and 500 cells per 60 mm dish) in triplicate dishes per condition, in 4 ml growth 

medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum, and 

incubated at 37 C in a humidified atmosphere of 5% CO2 + air.

2. Twenty-four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle 

(negative control) are added 1:1 (v/v) to the seeding medium; cells are incubated for 24 hr at 37 

C.

3. Final concentrations of test chemical to be tested initially will cover a dose range from 30-0.1 

μg/ml in approximate half-log dilutions. However, if desirable, the highest dose to be tested will 

be determined by the limits of solubility of the test agent.

4. After a 24 hour exposure period, the treatment medium is withdrawn; cells are washed once 

with Hanks’ balanced salt solution (pH 7.0) and resupplied with 5 ml fresh growth medium.
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5. The medium in all plates is replaced every 3-4 days for the duration of the assay.

6. The plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and scored 7-10 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded) and relative colony-forming efficiency (RCE) (i.e. number 

of colonies in chemically-treated dishes relative to the number of colonies in solvent-treated 

controls).

7. The approximate LD50 is determined from the relative survival data. The LD50 is that dose 

of test chemical which is lethal to 50% of the target cells.

APPENDIX B

Standard Protocol for the Mutagenesis of BALB 3T3 Cl. A31 Mouse Cells at the OuabainResistance Locus (OuaR

) in the Absence of Exogenous Metabolic Activation: Plate Assay

1. BALB 3T3 Cl. A31 cells from sub-confluent stock cultures are seeded at 3 different cell 

densities (250-500 cells per 60 mm dish, in triplicate dishes per condition, for cytotoxicity tests; 

1 x 104 cells per 60 mm dish, 15 dishes per condition, for mutation tests) in 4 ml growth medium 

(basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum, and incubated at 37C 

in a humidified atmosphere of 5% CO2 + air.

2. Twenty-four hours later, 2x concentrations of potential mutagen (i.e. test chemical (3 doses), 

or positive control chemical) or solvent vehicle (negative control) are added 1:1 (v/v) to the 

seeding medium; cells are incubated for 24 hr at 37 C. The doses of test chemical used shall be 

based upon the results obtained in a preliminary cytotoxicity assay, and shall include the 

approximate LD50, one dose above and one dose below the LD50. If an agent is non-toxic, then

the highest dose that will be used will be a function of the limits of solubility of the test agent.

3. Treatment medium is withdrawn; cells are washed once with Hanks’ balanced salt solution 

(pH 7.0) and resupplied with 5 ml fresh growth medium.

4. The medium in the cytotoxicity plates is replaced every 3 days for 7-10 days, after which the 

cells are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and scored for relative planting 

efficiency (RPE) (i.e. number of surviving colonies per number of cells seeded). These data 

establish the cytotoxic effects of the 24 hr treatment with the test chemical.

5. The medium in the mutagenesis test plates is replaced every 3 days until day 6 of the assay 

(the period of fixation and expression of the mutagenic event.)

6. On day 6 of the assay, the cells in two representative mutagenesis test plates are counted to 

establish the number of cells at risk (CAR) to the selective agent (which discriminates mutant 

cells).

7. The medium in the remaining mutagenesis test plates is replaced with fresh growth medium 

containing 1mM ouabain (a synthetic steroid which serves as the selective agent).
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8. The plates are incubated for 12 days at 37 C. Thereafter the growth medium containing 1 mM 

ouabain is changed once each week for 3-4 weeks.

9. Cells are fixed, stained and scored for the development of mutant ouabain-resistant colonies.

10. Mutation frequency (MF) data can be expressed in one of two ways: either (a) as the number 

of ouabain-resistant mutants per number of cells at risk to the selective agent, or (b) as the total 

number of ouabain-resistant mutants per condition.

RESULTS 

Genotoxicity result: Positive

Cytotoxic concentration (if applicable): Relative survival at 1:10000, 1:15000, 1:2000, 1:3000 

(0.1, 0.07, 0.05, 0.33 μl/ml) was 0.69%, 21.40%, 30.00% and 42.8%, respectively.

Results remarks: In the preliminary study, a dose response was not observed due to the 

extreme toxicity of DETDA to the target cells. The test agent was 100% cytotoxic at all of the 

concentrations tested although a few single cells were observed at the 1:1000 dilution; however, 

no colony formation was observed. Compound DETDA precipitated heavily at all of the 

concentrations tested.

In an additional preliminary study using revised dilutions, DETDA was observed to be 100% 

cytotoxic at the 1:1000, 1:2000 and 1:3000 dilutions. Although cell survival and colony 

formation was observed at the 1:5000 dilution, the relative survival of the target cells at this 

concentration was judged to be too low to be used in the transformation assay.

In the cytotoxicity study which accompanied the mutagenesis assay, a dose response was 

observed. Although DETDA was virtually 100% cytotoxic at the 1:10000 dilution (0.69% 

relative survival), the (%) relative survival at the 1:15000, 1:20000, and 1:30000 dilutions was 

found to be acceptable for the completion of the mutagenesis assay. The approximate LD40 was 

found to be at the 1:30000 dilution of compound DETDA.

In the mutagenesis assay, DETDA was effective in inducing ouabin-resistant colonies at all of 

the doses tested. The numbers of ouabain-resistant colonies observed at the 1:10,000 and 

1:15,000 dilutions are statistically significant relative to the medium control. However, the 

number of ouabain-resistant colonies observed at the 1:20,000 and 1:30,000 dilutions are not 

statistically significant relative to the medium control. No spontaneous mutant colonies were 

induced among cells in the medium control (negative control). MNNG (the positive control 

chemical) induced 20 ouabain-resistant colonies per 6.5 x 106 cells at risk..

CONCLUSIONS

Based on the results and discussion the following conclusions were made: (1) DETDA was 

found to be extremely cytotoxic to the BALB/3T3 Cl. A31 calls. (2) DETDA precipitated in the 

complete medium at all dilutions tested <1:10000. (3) A dose response was observed only 
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among dilutions tested >1:10000. (4) Under the conditions of the bioassay, the test chemical 

DETDA was demonstrated to be positive in producing ouabain-resistant colonies at all 

concentrations tested. Relative to the medium control (negative control), the numbers of mutant 

colonies induced by the 1:10000 and 1:15000 dilutions of test chemical were statistically 

significant (p<0.05). The numbers of mutant colonies observed at the 1:20000 and 1:30000 

dilutions of test chemical were not statistically significant (p>0.05) relative to the medium 

control (negative control).

STUDY RELIABILITY

[3] Not reliable. The study did not indicate the test guideline(s) followed, GLP compliance, and 

test substance purity and composition. Additionally, the cells that were tested, BALB/3TC 

Cl.A31 mouse cells, are not named in OPPTS 870.5300 as one of the cell lines suitable for 

detection of gene mutations in mammalian cells and the test substance was not tested in the 

presence of metabolic activation.

REFERENCES

Schechtman, L., Beard, S., and Fabrizio, D. 1978. Activity of DETA in in vitro mammalian cell 

point mutation assay. Performing Laboratory: Microbiological Associates. Bethesda, MD. 

Sponsor: Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 2011. 

Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194. 
Final HPV2 Chemical Data Adequacy Review (May 2012 Update) CAS No. 68479-98-1

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C-7. Genetic Toxicity – Chromosomal Aberrations: Test Requirement (E2) under Final 

HPV2 Test Rule Dated January 7, 2011

Summary of endpoint: Adequate

Study 1: Chromosomal aberrations study in human lymphocytes (Durward, 2000)

Title: LZ1224.20: Chromosome Aberration Test in Human Lymphocytes in vitro

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonyms: 

Diethyltoluenediamine (DETDA), DETDA 80; Purity: 98.4%; Composition of test material: 

77.6% 3,5-diethyltoluene-2,4-diamine (GC), 20.8% 3,5-diethyltoluene-2,6-diamine (GC); 

Impurities: 0.4% ethyl toluenediamine (GC), < 0.0150% toluenediamine (GS), 0.02% (w/w) 

water

Remarks: Clear liquid; Batch No. 2235

METHOD

Method/guideline followed: OECD 473 

Test type: In vitro mammalian chromosomal aberration test

GLP compliant?: Yes

Year study performed: 1999-2000

Test species: Human lymphocytes

Metabolic activation: With and without

Concentrations tested: 0 (negative control); 0 (DMSO vehicle control); Preliminary toxicity 

test: 7 to 1800 μg/mL (with and without S9); Experiment 1: 450 to 1800 μg/mL (with and 

without S9); Experiment 2: 112.5 to 337.5 μg/mL (without S9), 450 to 1800 μg/mL (with S9); 

Experiment 3: 1400 to 1800 μg/mL (with S9)

Statistical methods used: Fisher’s exact test, chi-squared test

Test method/conditions remarks: Duplicate lymphocyte cultures (A and B) were established 

for each concentration level. 
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A preliminary toxicity test was performed on cell cultures using a 4-hour exposure with and 

without metabolic activation followed by a 20-hour recovery period and a continuous exposure 

of 24 hours without metabolic activation. The concentration range of test material used was 7 to 

1800 μg/mL. Using a qualitative microscopic evaluation of the slides prepared from the 

treatment cultures, appropriate concentration levels were selected for mitotic index evaluation. 

Mitotic index data were used to estimate the test material toxicity and select concentration levels 

for the main study in which vehicle and positive controls were used in parallel with the test 

material. 

Experiment 1: Four-hour exposures to DETDA were conducted with or without S9, followed by 

20-hour culture in treatment-free media prior to cell harvest. After mitotic index determination, 

the concentration levels selected for metaphase analysis were: 450, 900, and 1800 μg/mL.

Experiment 2: Twenty-four hour continuous exposures to DETDA without S9 were conducted 

prior to cell harvest. After mitotic index determination, the concentration levels selected for 

metaphase analysis were: 112.5, 225, and 337.5 μg/mL. Four-hour exposure to DETDA with S9 

was conducted, followed by 20-hour culture in treatment-free media prior to cell harvest. After 

mitotic index determination, the concentration levels selected for metaphase analysis were: 450, 

900, 1350, and 1800 μg/mL. 

Experiment 3: Four-hour exposures to DETDA with S9 (either 2% or 5% final concentration) 

were conducted, followed by 20-hour culture in treatment-free media prior to cell harvest. After 

mitotic index determination, the concentration levels selected for metaphase analysis were: 1400

and 1800 μg/mL.

Cell Harvest: Mitosis was arrested by addition of demecolcine two hours before the required 

harvest time. The cells were resuspended in hypotonic KCl, fixed in methanol/glacial acetic acid, 

and stained in Gurr’s Giemsa. The slides were screened microscopically for the quality of the 

metaphase (i.e. well spread, undamaged, perfectly stained) and were coded randomly. A total of 

2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and 

expressed as the mitotic index and as a percentage of the vehicle control value. A positive 

response was recorded for a particular treatment if the percentage of cells with aberration, 

excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a 

clear concentration relationship. For modest increases in aberration frequency, a concentrationresponse relationship was required and an appropriate statistical test was applied in order to 

record a positive response. The frequency of cells with aberrations (both including and 

excluding gaps) and the frequency of polyploidy cells were compared, where necessary, with the 

concurrent vehicle control value using Fisher’s exact and chi-squared tests.

Vehicle Used?: Yes

Vehicle Name: DMSO

Positive Control Substances: Without metabolic activation: ethylmethanesulphonate 

(Experiment 1) and mitomycin C (Experiment 2). With metabolic activation: 

cyclophosphamide. 
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RESULTS

Genotoxicity result: Negative without metabolic activation; equivocal with metabolic 

activation

Cytotoxic concentration (if applicable): Not clearly defined; see “results remarks” section.

Results remarks: 

Preliminary Toxicity Test: DETDA induced toxicity in all three treatment groups: (1) 4-hour 

treatment, 20-hour recovery without S9; (2) 4-hour treatment, 20-hour recovery with S9; (3) 24-

hour treatment without S9. The response was more pronounced in the continuous exposure 

cultures without S9. No DETDA precipitate was observed. Microscopic assessment of the 

slides prepared from the treatment cultures showed that metaphase cells were present up to the 

10 mM limit concentration of DETDA (1800 μg/mL) in the pulse treatment with and without S9, 

and up to 450 μg/mL in the 24-hour continuous treatment without S9. 

Experiment 1: DETDA did not induce any statistically significant increases in the frequency of 

cells with chromosomal aberrations either in the absence or presence of metabolic activation. 

However, a concentration-related trend was observed when gap-type aberrations were taken into 

account. DETDA induced a significant increase in the numbers of polyploid cells in the absence 

of S9 at 1800 μg/mL, and in the presence of S9 at 900 μg/mL. 

A concentration-related inhibition of mitotic index was observed such that 42% mitotic 

inhibition was achieved at 1800 μg/mL in the absence S9. In the presence of S9, the 

concentration-related response was not pronounced and only 23% mitotic inhibition was 

achieved at the maximum concentration level, 1800 μg/mL.

Experiment 2: DETDA induced a statistically significant concentration-response increase in the 

frequency of cells with chromosome aberrations in the presence of S9. A similar response was 

not observed in Experiment 1. However, the final S9 concentration was increased from 1% to 

2%, which may explain the different qualitative responses seen in the two experiments. DETDA 

induced a significant increase in the number of polyploid cells at 900 μg/mL in the presence of 

S9, confirming the response seen in Experiment 1.

DETDA induced a concentration-related inhibition of mitotic index such that a near optimum of 

52% mitotic inhibition was achieved at 337.5 μg/mL in the absence of S9. The 87% inhibition 

observed at 450 μg/mL (with S9) was considered to be too excessive for that concentration level 

to be included in the metaphase analysis. In the presence of S9, the concentration-related 

response was less clear; in the concentration levels that were assessed, mitotic inhibition was 

induced to a maximum of 41% of the control value at 1800 μg/mL. 

Experiment 3: DETDA did not induce any statistically significant increases in the frequency of 

cells with chromosome aberrations excluding gap-type aberrations either in the 2 or 5% final S9 

concentration treatment groups. However a significant increase was seen when gap-type 

aberrations were included at 1800 μg/mL in the 5% S9 treatment group. The response seen in 
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Experiment 2 was not reproduced in the 2% S9 treatment group, and only a gap-type response 

was observed in the 5% final S9 concentration group. However, an unavoidable difference 

between Experiment 2 and Experiment 3 was that a different batch of S9 was used with a slightly 

reduced protein content. DETDA induced a significant increase in the numbers of polyploidy 

cells at the 1800 μg/mL concentration level in the 2% S9 concentration treatment group; 

confirming the responses seen in both Experiment 1 and 2. The polyploidy cells observed were 

primarily endoreduplicated cells in all three experiments. 

DETDA induced a concentration-related reduction in mitotic index using both S9 concentrations, 

such that 45% mitotic inhibition was achieved at 1800 μg/mL in 2% S9 cultures, and 52% 

mitotic inhibition was seen at 1800 μg/mL in the 5% S9 cultures.

The positive control treatments performed as expected. All the vehicle control cultures had 

frequencies of cells with chromosome aberrations within the expected range. 

CONCLUSIONS

The test material induced no statistically significant increase in the frequency of cells with 

aberrations (excluding gaps) in the first experiment. However, in the second experiment, an 

increase was observed with metabolic activation (2% S9), which was not clearly concentrationrelated. This response was not reproduced in the third experiment using 2% or 5% S9, but 

different S9 preparations were used in the second and third experiments. A small statistically 

significant increase in the frequency of cells with aberrations including gaps was also observed 

in the 5% S9 group of the third experiment. Small increases in polyploid cell frequency, both in 

the absence and presence of S9, were also noted. There was equivocal evidence for a clastogenic 

effect on human lymphocytes of DETDA in the presence of metabolic activation, but no 

evidence for a clastogenic effect in the absence of metabolic activation.

STUDY RELIABILITY

[2] Reliable with restrictions. The report was supplied as an IUCLID summary and important 

details were missing including quantitative data on the number of cells with chromosomal 

aberrations and types of chromosomal aberrations for control and treated cultures. 

REFERENCE

Durward, R. 2000. LZ1224.20: Chromosome Aberration Test in Human Lumphocytes in Vitro, 

SPL Project Number: 102/300. Study No. 2077. Performing Laboratory: SafePharm 

Laboratories, Ltd. Shardlow, Derbyshire. Sponsor: Lonza Limited, Basle, Switzerland. (Cited 

as Durwood [2000] in summary submitted by Albemarle and Lonza on March 1, 2012)
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Study 2: Micronucleus study in mice (Durward and Nolan, 2000)

Title: LZ1224.20: Micronucleus Test in the Mouse

The study is: Adequate Not Adequate

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonyms: 

Diethyltoluenediamine (DETDA), DETDA 80; Purity: 98.4%; Composition of test material: 

77.6% 3,5-diethyltoluene-2,4-diamine (GC), 20.8% 3,5-diethyltoluene-2,6-diamine (GC); 

Impurities: 0.4% ethyl toluenediamine (GC), < 0.0150% toluenediamine (GS), 0.02% (w/w) 

water

Remarks: Amber slightly viscous liquid; Batch No. 2235

METHOD

Method/guideline followed: OECD 474

Test type: Mouse erythrocyte micronucleus test

GLP compliant?: Yes

Year study performed: 2000

Test species: ICR mice (5-8 weeks old, 25-30 g both sexes combined)

Number of animals/sex/dose: 2/sex/dose (preliminary range-finding toxicity test); 7 males/dose 

(micronucleus test)

Route of administration: Oral gavage

Vehicle: Arachis oil

Metabolic activation: N/A

Doses tested: 0 (DMSO vehicle control), 125, 250, or 500 mg/kg-bw test material; 50 mg/kgbw cyclophosphamide (positive control).

Statistical methods used: Student’s “t” test, One way Analysis of Variance

Test method/conditions remarks: In a preliminary dose-range study, groups of 2 male and 2 

female mice were dosed with DETDA at concentrations of 300, 450, or 600 mg/kg-bw. Based 

on the results of the dose-range-finding study, the DETDA dose levels selected for the 

micronucleus test were 125, 250, and 500 mg/kg-bw. 
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In the micronucleus test, DETDA was administered via a single oral gavage dose to groups of 7 

male mice at doses of 125, 250, or 500 mg/kg-bw. One group of mice from each dose level was 

sacrificed 24 hours following treatment, and a second group dosed at 500 mg/kg-bw at 48 hours. 

Cyclophosphamide served as a positive control (50 mg/kg-bw) for a group of 7 mice, and the 

vehicle, arachis oil, served as the negative control for two groups of mice (n = 7/group). The 

vehicle controls were sacrificed 24 or 48 hours following dosing and positive control group 

animals were sacrificed 24 hours following dosing. Immediately following sacrifice, marrow 

was aspirated from the femurs and used to prepare microscopic slides for evaluation following 

May-Grunwald/Giemsa staining.

Incidences of micronucleated cells were determined in 2000 polychromatic erythrocytes (PCE) 

per animal. In addition, incidences of normochromatic erythrocytes (NCE) per 1000 

erythrocytes were determined and the NCE also were scored for incidences of micronuclei. The 

PCE/NCE ratio was calculated together with appropriate group mean values and standard 

deviation. A comparison was made between the number of micronucleated PCE occurring in 

each of the test material groups and the number occurring in the corresponding vehicle control 

group. Results were analyzed according to the Student’s “t” test and any significant results were 

confirmed using the one way analysis of variance. 

RESULTS

Genotoxicity result: Negative

Cytotoxic dose: Not indicated

Results remarks: Preliminary Range-Finding Toxicity Test: Premature death occurred at 600 

mg/kg-bw and clinical signs were observed at and above 300 mg/kg-bw, as follows: lethargy, 

hunched posture, ptosis, decreased respiratory rate, labored respiration, ataxia, and splayed gait. 

DETDA showed no marked difference in its toxicity to male or female mice. Therefore, males 

were used for the definitive study. Adequate evidence of DETDA toxicity was demonstrated via 

the oral route of administration to warrant the same route in the main study. The maximum 

tolerated dose of DETDA, 500 mg/kg-bw, was selected for use in the main study, with 250 and 

125 mg/kg-bw as the lower dose levels. 

Micronucleus Test: There were no premature deaths, but clinical signs including hunched 

posture, lethargy, and ptosis, were observed at ≥ 125 mg/kg-bw in both the 24- and 48-hour 

groups. The presence of clinical signs indicated that systemic absorption had occurred. There 

were no statistically significant decreases in the PCE/NCE ratio in the 24- or 48-hour DETDA 

groups compared to the concurrent vehicle control groups. There were no statistically significant 

increases in the frequency of micronucleated PCE in any of the DETDA dose groups when 

compared to their concurrent vehicle control groups. 

The positive control performed as expected. 
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CONCLUSIONS

The test substance did not induce micronuclei in bone marrow polychromatic erythrocytes of 

mice treated by a single oral gavage dose (125, 250 or 500 mg/kg-bw) of the test substance.

STUDY RELIABILITY

[2] Reliable with restrictions. The report was supplied as a summary and important details were 

missing including mean and standard deviations of micronucleated PCE for each dose group and 

PCE/NCE ratios by dose group. 

REFERENCE

Durward, R. and Nolan, S. 2000. LZ1224.20: Micronucleus Test in the Mouse, SPL Project 

Number: 102/340. Study No. 2080. Performing Laboratory: SafePharm Laboratories, Ltd. 

Shardlow, Derbyshire. Sponsor: Lonza Limited, Basle, Switzerland. (Cited as Durwood and 

Nolan [2000] in summary submitted by Albemarle and Lonza on March 1, 2012)
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Study 3: Micronucleus study in mice (Naismith and Matthews, 1979)

Title: Micronucleus Test (MNT)

This study is: Adequate Not Adequate

Important details were missing from the study including purity and composition of the test 

substance and cytotoxicity. Additionally, this study is not reliable for the following reasons: (1) 

The study tested only two doses whereas CFR 799.9539 stipulates the testing of three doses that 

cover a range from the maximum to little or no toxicity; (2) The study used 4 animals/sex/dose 

whereas CFR 799.9539 stipulates the testing of at least 5 analyzable animals/sex/group; (3) The 

study sacrificed animals at 6 hours after the second of two doses whereas CFR 799.9539 

stipulates that sacrifice for bone marrow sampling between 18 and 24 hours following the final 

treatment; and (4) The study reported the number of micronuclei per 1000 polychromatic 

erythrocytes whereas CFR 799.9539 stipulates the scoring of at least 2000/animal. 

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediame (DETDA); Purity not specified

Remarks: Dark brown liquid; Sample No. 10121

METHOD

Method/guideline followed: Standard Operating Procedure PH-309

Test type: Mouse bone marrow micronucleus test

GLP compliant?: Yes

Year study performed: 1979

Test species: CF-1 mice (7-12 weeks old, 22-26 g males, 22-25 g females)

Number of animals/sex/dose: 4

Route of administration: Intraperitoneal injection

Vehicle: 0.25% Methylcellulose

Metabolic activation: N/A

Doses tested: 0 (vehicle control), 50 and 100 mg/kg-bw

Statistical methods used: Student’s “t” test
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Test method/conditions remarks: In a preliminary dose-range study, DETDA was dosed i.p. 

once daily for two days to groups of ten mice at concentrations of 500, 250, 150, 100 and 50 

mg/kg. Based on the results of the dose-range-finding study, the dose levels for DETDA in the 

Micronucleus Test were chosen at 100 mg/kg and 50 mg/kg. 

In the Micronucleus Test, DETDA was administered via intraperitoneal injection once daily for 2 

days to 4 female and 4 male mice at doses of 50 or 100 mg/kg-bw. Triethylenemelamine served 

as a positive control (0.5 mg/kg-bw), and the vehicle, 0.25% methylcellulose (20 mL/kg-bw) as 

the negative control. The positive and negative controls were administered to two groups of 8 

mice. Mice were sacrificed 6 hours after the second dose, marrow was aspirated from the femurs 

and used to prepare microscopic slides for evaluation.

The slides are screened for good preparation, i.e. well spread, undamaged, perfectly stained. One 

thousand (1000) polychromatic erythrocytes are counted for the presence of micronuclei. The 

frequency of micronucleated cells is expressed as percent micronucleated cells versus total 

polychromatic erythrocytes. Roughly 8% of the cells are erythrocytes and half are 

polychromatic. The slides are examined using the oil immersion objective of an American 

Optical Microstar® Binocular Microscope. Results are analyzed according to the Student’s “t” 

test. 

RESULTS

Preliminary Study: The 500 mg/kg dose group exhibited severe ataxia, passivity, salivation, loss 

of righting and twitches. Seven mice died within one hour of dosing. The remaining three mice 

exhibited identical symptomatology after the second dose, and all mice died within one hour 

post-dose. 

The 250 mg/kg dose group exhibited ataxia, complete body drop, ptosis, tonic behavior, brief 

loss of righting, decreased body tone and decreased spontaneous activity. All symptoms were 

very pronounced. A 2 – 6 gram per mouse weight loss was noted after the second dose with one 

mouse dying one hour after the second dose. 

The 150 mg/kg dose group exhibited slight ataxia, complete body drop, decreased body tone, 

passivity, ptosis, depression and decreased spontaneous activity. No deaths occurred. 

The 100 mg/kg dose group exhibited body drop, loss of body tone, ptosis, depression, abnormal 

gait, and decreased spontaneous activity. No deaths occurred.

The 50 mg/kg dose group exhibited decreased spontaneous activity slight loss of body tone and 

depression. No deaths occurred.

Micronucleus Test: DETDA was negative for the induction of micronuclei, based on the inability 

of the test article to produce a statistically significant increase in the number of micronuclei per 

1000 polychromatic erythrocytes in the treated versus the control groups. 

Genotoxicity result: Negative
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Cytotoxic dose: Not indicated

Results remarks: The positive control performed as expected. As per the sponsor’s request, 

slides from the Micronucleus Test on DETDA were rescored in 1984 according to present 

criteria. The slides were first counted in 1979, prior to changes in the criteria for scoring 

micronuclei.

CONCLUSIONS

The test substance did not induce micronuclei in bone marrow erythrocytes of mice treated by 

two consecutive daily intraperitoneal injections at either dose (50 or 100 mg/kg-bw).

STUDY RELIABILITY

[3] Not reliable. Important details were missing from the study including purity and composition 

of the test substance and cytotoxicity. Additionally, this study is not reliable for the following 

reasons: (1) The study tested only two doses whereas CFR 799.9539 stipulates the testing of 

three doses that cover a range from the maximum to little or no toxicity; (2) The study used 4 

animals/sex/dose whereas CFR 799.9539 stipulates the testing of at least 5 analyzable 

animals/sex/group; (3) The study sacrificed animals at 6 hours after the second of two doses 

whereas CFR 799.9539 stipulates that sacrifice for bone marrow sampling between 18 and 24 

hours following the final treatment; and (4) The study reported the number of micronuclei per 

1000 polychromatic erythrocytes whereas CFR 799.9539 stipulates the scoring of at least 

2000/animal.

REFERENCE

Naismith, R. and Matthews, R. 1979. Micronucleus Test (MNT). Study No. PH-309-ET-004-

79. Performing Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl 

Corporation, Baton Rouge, LA. 
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C-8. Reproductive Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011

Summary of endpoint: Not adequate

No studies were submitted for this endpoint.
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C-9. Developmental Toxicity: Test Requirement (F1) under Final HPV2 Test Rule Dated 

January 7, 2011

Summary of endpoint: Not adequate

No studies were submitted for this endpoint.
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D. SUPPLEMENTAL DATA (NON-SIDS ENDPOINTS)

D-1. Genetic Toxicity – Other 

Study 1: Dominant lethal assay in rats (Schechtman et al., 1979)

Title: Activity of T1363 (otherwise known as diethyltoluenediamine) in the dominant lethal 

assay in rodents for mutagenicity

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA) 

Remarks: T1363; brown liquid.

METHOD

Method/guideline followed: Not indicated

Test type: Rodent dominant lethal assay

GLP compliant?: Not indicated

Year study performed: 1979

Test species: Sprague Dawley rats, males (n=10/group); at start of test initiation, rats were 8-10 

weeks old and weighed 225-260 g (males) or 185-215 g (females).

Route of administration: Oral - gavage

Vehicle: Corn oil

Metabolic activation: Not applicable

Concentrations tested: 0, 0.7, 7 or 70 mg/kg-bw

Statistical methods used: T-test, Chi-square, Analysis of Regression, Analysis of Linear Trend, 

Analysis of Variance, Probit Analysis, and Freeman-Tukey test.

Test method/conditions remarks: The dominant lethal assay was performed by sequentially 

mating males, following chemical administration, with groups of untreated females over the 

duration of the spermatogenic cycle. Weeks 1 and 2 of sequential mating of treated males with 

pairs of untreated females served to identify action of postmeiotic stages of spermatogenesis; 

three and four on meiotic stages, and weeks five through eight on premeiotic stages. Positive 

(triethylene melamine) and negative controls (corn oil) were run concurrently. Dominant lethal 

mutations were measured directly by enumeration of fetal deaths and indirectly by pre-
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implantation losses, as measured by reduction in the number of total implants in test groups as 

compared with control females. 

In this study, three groups (n = 10/group) of male Sprague-Dawley rats were administered 5 

treatments of DETDA via gavage in corn oil at 0, 0.7, 7 or 70 mg/kg-bw. 0.5 mg/kg-bw of the 

positive control was administered via a single injection, and 4 mL/kg of the negative control was 

administered via gavage. The following week, each male was mated with 2 females from 

Monday through Friday. On the subsequent Monday, the male was mated with 2 new females 

and the process repeated until each male had been mated for 7 weeks with 2 new females/wk. 

Fourteen days from the mid-week of mating, females were sacrificed and the reproductive tract 

examined. Nine parameters were analyzed: fertility index, total implantations per pregnant 

female, corpora lutea per pregnant female, pre-implantation losses per pregnant female, dead 

implants per pregnant female, proportion of pregnant females with one or more dead implants, 

proportion of pregnant females with two or more dead implants, dead implants per total implants, 

and live implants per pregnant female. Appropriate statistical methods were used to compare 

control and treated animals.

RESULTS 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not applicable

Results remarks: The negative controls were within the normal range of values for this species 

and strain as observed in the test laboratory. The positive control exhibited its usual severe 

genetic damage between weeks 2 and 7 as shown by greater pre-implantation losses, increased 

number of dead implants per female, decreased number of live implants per female, and more 

females with one or more as well as two or more dead implants.

The test compound exhibited minimal activity in total implantations, pre-implantation losses, 

number of dead implants per total implants and number of live implants per pregnant female. No 

statistically significant dose response was observed. 

CONCLUSIONS

It was concluded DETDA exhibited little or no mutagenic activity in the dominant lethal test.

STUDY RELIABILITY

[3] Not reliable. Test guideline and GLP compliance were not indicated and a rationale for dose 

selection was not presented. Additionally, evidence was not provided, as stipulated by the 

OPPTS 870.5450 guideline for the Rodent Dominant Lethal Assay, that the highest dose 

produced signs of toxicity (e.g. slightly reduced fertility or slightly reduced body weight).
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REFERENCE

Schechtman, L., Putman, D., Pannar, A., and Sinsky, P. 1979. Activity of T1363 (otherwise 

known as diethyltoluenediamine) in the dominant lethal assay in rodents for mutagenicity. 

Performing Laboratory: Microbiological Associates, Bethesda, MD. Sponsor: Ethyl 

Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 2011. Letter to EPA DCO 

dated May 4, 2011. OPPT-2007-0531, MR #335194.
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Study 2: DNA repair test in primary rat hepatocytes (Johnson and Mulholland, 1984)

Title: Rat hepatocyte primary culture/DNA repair test

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; referred to as 

diethyltoluenediamine (DETDA); purity not specified

 

Remarks: Slightly yellow viscous liquid

METHOD

Method/guideline followed: Protocol- ETTOX 028

Test type: Primary rat hepatocyte DNA repair test

GLP compliant?: Yes

Year study performed: 1984-1985

Test species: Primary rat hepatocytes from adult male Fischer-344 rats (150-300 grams)

Metabolic activation: None

Concentrations tested: 0 (DMSO solvent control), 0 (untreated control), 0.05, 0.1, 0.5, 1, 5, 10, 

50, 100, 500 and 1000 μg/well in 2 mL of media.

Statistical methods used: Chi square analysis 

Test method/conditions remarks: The test substance was dissolved in DMSO. DMSO was 

used as a vehicle control and 2-acetamidofluorene (2AAF) was the positive control.

Freshly isolated rat hepatocytes were treated with 20 μl of DETDA at 0.05, 0.1, 0.5, 1, 5, 10, 50, 

100, 500 and 1000 μg/well in 2 mL of media. In addition, a DMSO group, an untreated control, 

and 2-acetoamido fluorine (2AAF) at a concentration of 10-6 M, were evaluated concurrently. 

Isolation of Adult Rat Hepatocytes: The protocol for isolating adult rat hepatocytes is a 

modification of the method described by Williams (1978).

Perfusion Media: 

Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029.

Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029.
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Surgical and Perfusion Procedure: Male Fischer rats are anesthetized with Sodium Nembutal by 

intraperitoneal injection. Following anesthetization, the liver is perfused and removed following 

the procedure outlined in SOP # ETTOX 029.

Cell harvesting: The liver is excised and the hepatocytes are harvested according to SOP 

# ETTOX 029.

Dosage Selection: The highest dose will be 5% solution or the highest soluble concentration of 

the test article in the appropriate solvent. The remaining nine doses will be half-log dilutions 

from the 5% solutions.

Test Protocol: Hepatocytes are treated with the test article, fixed onto microscope cover slides, 

stained, dipped, and developed according to SOP # ETTOX 029.

Data Quantification: Unscheduled DNA repair synthesis, evidenced by a net increase in black 

silver grains in the nucleus, is quantified by determining nuclear and background grain counts of 

25 cells/slide or as many cells as possible (up to 25) in the presence of toxicity using an 

automatic colony counter with a microscopic attachment. The test compound is reported as 

positive or equivocal if there is a dose-related increase of mean net nuclear grain counts, a mean 

net nuclear grain count of 5 over background is consistently observed, and/or there is a 

statistically significant dose related increase over background of cells with net nuclear counts 

greater than zero.

RESULTS

The highest dose scored was 1000 μg/well. Unscheduled DNA repair synthesis was quantified 

by a net nuclear increase of black silver grains for 25 cells/slide. This value was determined by 

taking a nuclear count and the average of three adjacent cytoplasmic counts.

DETDA did not cause an increase in mean net nuclear counts over DMSO at any dose levels 

counted (1000, 500 and 100 μg/well). A Chi square analysis was done to compare treated cells 

to untreated controls. The number of cells with net nuclear grain counts greater than zero of all 

scored dose levels of DETDA was not significantly increased statistically over DMSO.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Slight cytotoxicity was observed at 1000 μg/well.

Results remarks: The vehicle (DMSO) and positive control net nuclear grain counts were 

acceptable. Color and appearance of the test article did not change from the time of receipt to 

the time of use. 

CONCLUSIONS

DETDA was not genetically active in the Rat Hepatocyte Primary Culture/DNA Repair Test 

according to Protocol – ETTOX 028.
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STUDY RELIABILITY

[2] Reliable with restrictions. Purity of test substance was not provided. Additionally, the study 

author did not provide analytical data to verify actual concentrations used in the assay.

REFERENCES

Johnson, D. and Mulholland, A. 1984. Rat Hepatocyte Primary Culture/DNA Repair Test. 

Diethyltoluenediamine. DETDA. Study No. HPC 023 - #073. Performing Laboratory: Genetic 

Toxicology Laboratory. Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 3: DNA repair test in primary rat hepatocytes (Johnson and Mulholland, 1986)

Title: Rat hepatocyte primary culture/DNA repair test

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Referred to as 

DETDA-OBG

 

Remarks: Lot #5868-1; Amber liquid; Composition: 0.7% METDA; 78.2% 2,4-DETDA; 

20.3% 2,6-DETDA; 0.8% other isomers, trialkylated toluenediaminea

METHOD

Method/guideline followed: Protocol- ETTOX 028

Test type: Primary rat hepatocyte DNA repair test

GLP compliant?: Yes

Year study performed: 1986

Test species: Primary rat hepatocytes from adult male Fischer-344 rats (150-300 grams)

Metabolic activation: None

Concentrations tested: 0 (DMSO solvent control), 0 (untreated control), 0.05, 0.1, 0.5, 1, 5, 10, 

50, 100, 500 and 1000 μg/well in 2 mL of media

Statistical methods used: Chi square analysis

Test method/conditions remarks: The test substance was dissolved in DMSO. DMSO was 

used as a vehicle control and 2-acetamidofluorene was the positive control.

Freshly isolated rat hepatocytes were treated with 20 μl of DETDA-OBG at 0.05, 0.1, 0.5, 1, 5, 

10, 50, 100, 500 and 1000 μg/well in 2 mL of media. In addition, a DMSO group, an untreated 

control, and 2-Acetoamido fluorine (2AAF) at a concentration of 10-6

M, were evaluated 

concurrently. 

Isolation of Adult Rat Hepatocytes: The protocol for isolating adult rat hepatocytes is a 

modification of the method described by Williams (1978).

Perfusion Media: 

Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029.

Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029.

 a Composition taken from Mulholland, 1986
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Surgical and Perfusion Procedure: Male Fischer rats are anesthetized with Sodium Nembutal by 

intraperitoneal injection. Following anesthetization, the liver is perfused and removed following 

the procedure outlined in SOP # ETTOX 029.

Cell harvesting: The liver is excised and the hepatocytes are harvested according to SOP # 

ETTOX 029.

Dosage Selection: The highest dose will be 5% solution or the highest soluble concentration of 

the test article in the appropriate solvent. The remaining nine doses will be half-log dilutions 

from the 5% solutions.

Test Protocol: Hepatocytes are treated with the test article, fixed onto microscope coverslides, 

stained, dipped, and developed according to SOP # ETTOX 029.

Data Quantification: Unscheduled DNA repair synthesis, evidenced by a net increase in black 

silver grains in the nucleus, is quantified by determining nuclear and background grain counts of 

25 cells/slide or as many cells as possible (up to 25) in the presence of toxicity using an 

automatic colony counter with a microscopic attachment. The test compound is reported as 

positive or equivocal if there is a dose-related increase of mean net nuclear grain counts, a mean 

net nuclear grain count of 5 over background is consistently observed, and/or there is a 

statistically significant dose related increase over background of cells with net nuclear counts 

greater than zero.

RESULTS

The highest dose scored was 5 μg/well. Unscheduled DNA repair synthesis was quantified by a 

net nuclear increase of black silver grains for 25 cells/slide. This value was determined by 

taking a nuclear count and the average of three adjacent cytoplasmic counts.

The results for test article DETDA-OBG were negative in the rat hepatocyte primary 

culture/DNA repair test under the conditions of this assay. These results are based upon the 

inability of DETDA-OBG to produce a mean grain count of five or greater than the vehicle 

control mean grain count and no statistically significant difference between the DETDA-OBG 

treated cells and the vehicle control in the number of cells with net nuclear grain counts greater 

than zero. No dose-related increase in net nuclear counts or cells greater than zero was 

demonstrated by DETDA-OBG. A Chi square analysis was done to compare treated cells to 

untreated controls.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 10, 50, 100, 500 and 1000 μg/well

Results remarks: The vehicle and positive control net nuclear grain counts were acceptable. 

Color and appearance of the test article did not change from the time of receipt to the time of use.
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CONCLUSIONS

DETDA-OBG was not genetically active in the rat hepatocyte primary culture/DNA repair test 

according to Protocol – ETTOX 028.

STUDY RELIABILITY

[2] Reliable with restrictions. The study author did not provide analytical data to verify actual 

concentrations used in the assay.

REFERENCES

Johnson, D. and Mulholland, A. 1986. Genetic Toxicology. Rat Hepatocyte Primary 

Culture/DNA Repair Test. DETDA-OBG. Study No. HPC 027-#099. Performing Laboratory: 

Genetic Toxicology Laboratory. Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 4: DNA repair test in primary rat hepatocytes (Johnson and Mulholland, 1986)

Title: Rat hepatocyte primary culture/DNA repair test

TEST SUBSTANCE 

Identity: 98.9% 3,5-Diethyltoluene-2,6-diamine, CAS No. 2095-01-4; Referred to as 2,6-

DETDA #2

Remarks: Lot #5712-71-6; Pale green crystalline solid; Composition: 98.9% 3,5-

diethyltoluene-2,6,diamine; 0.8% monoethyltoluenediamine; 0.3% other isomers.a

METHOD

Method/guideline followed: Protocol- ETTOX 028

Test type: Primary rat hepatocyte DNA repair test

GLP compliant?: Yes

Year study performed: 1986

Test species: Primary rat hepatocytes from adult male Fischer-344 rats (150-300 grams)

Metabolic activation: None

Concentrations tested: 0 (DMSO solvent control), 0 (untreated control), 0.05, 0.1, 0.5, 1, 5, 10, 

50, 100, 500 and 1000 μg/well in 2 L of medium

Statistical methods used: Not indicated

Test method/conditions remarks: The test substance was dissolved in DMSO. DMSO was 

used as a vehicle control and 2-acetamidofluorene was the positive control.

Freshly isolated rat hepatocytes were treated with 20 μl of 2,6-DETDA #2 at 0.05, 0.1, 0.5, 1, 5, 

10, 50, 100, 500 and 1000 μg/well in 2 mL of media. In addition, a DMSO group, an untreated 

control, and 2-Acetoamido fluorine (2AAF) at a concentration of 10-6

M, were evaluated 

concurrently. 

Isolation of Adult Rat Hepatocytes: The protocol for isolating adult rat hepatocytes is a 

modification of the method described by Williams (1978).

Perfusion Media: 

Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029.

 a Composition taken from Mulholland, 1986
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Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029.

Surgical and Perfusion Procedure: Male Fischer rats are anesthetized with Sodium Nembutal by 

intraperitoneal injection. Following anesthetization, the liver is perfused and removed following 

the procedure outlined in SOP # ETTOX 029.

Cell harvesting: The liver is excised and the hepatocytes are harvested according to SOP # 

ETTOX 029.

Dosage Selection: The highest dose will be 5% solution or the highest soluble concentration of 

the test article in the appropriate solvent. The remaining nine doses will be half-log dilutions 

from the 5% solutions.

Test Protocol: Hepatocytes are treated with the test article, fixed onto microscope coverslides, 

stained, dipped, and developed according to SOP # ETTOX 029.

Data Quantification: Unscheduled DNA repair synthesis, evidenced by a net increase in black 

silver grains in the nucleus, is quantified by determining nuclear and background grain counts of 

25 cells/slide or as many cells as possible (up to 25) in the presence of toxicity using an 

automatic colony counter with a microscopic attachment. The test compound is reported as 

positive or equivocal if there is a dose-related increase of mean net nuclear grain counts, a mean 

net nuclear grain count of 5 over background is consistently observed, and/or there is a 

statistically significant dose related increase over background of cells with net nuclear counts 

greater than zero.

RESULTS

The highest dose scored was 5 μg/well. Unscheduled DNA repair synthesis was quantified by a 

net nuclear increase of black silver grains for 25 cells/slide. This value was determined by 

taking a nuclear count and the average of three adjacent cytoplasmic counts.

The results for test article 2,6-DETDA #2 were negative in the Rat Hepatocyte Primary 

Culture/DNA Repair Test under the conditions of this assay. These results are based upon the 

inability of 2,6-DETDA #2 to produce a mean grain count of five or greater than the vehicle 

control mean grain count and no statistically significant difference between the 2,6-DETDA #2 

treated cells and the vehicle control in the number of cells with net nuclear grain counts greater 

than zero. No dose-related increase in net nuclear counts or cells greater than zero were 

demonstrated by 2,6-DETDA #2. 

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 10, 50, 100, 500 and 1000 μg/well

Results remarks: The vehicle and positive control net nuclear grain counts were acceptable. 

Color and appearance of the test article did not change from the time of receipt to the time of use.
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CONCLUSIONS

2,6-DETDA #2 was not genetically active in the Rat Hepatocyte Primary Culture/DNA Repair 

Test according to Protocol- Ettox 028.

STUDY RELIABILITY

[2] Reliable with restrictions. Missing study details included statistical methods used and 

analytical data to verify actual concentrations used in the assay.

REFERENCES

Johnson, D. and Mulholland, A. 1986. Genetic Toxicology. Rat Hepatocyte Primary 

Culture/DNA Repair Test. 2,6-DETDA #2. Study No. HPC 027-#100. Performing Laboratory: 

Genetic Toxicology Laboratory. Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 5: DNA repair test in E. coli (Schechtman et al., 1978)

Title: Activity of DETDA in a test for differential inhibition of repair deficient and repair 

competent strains of Escherichia coli: DNA repair

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Synonym:

Diethyltoluenediamine (DETDA); Purity not specified

 

Remarks: Referred to as “DETA”, identified as “Diethyltoluenediamine”, dark brown viscous 

liquid

METHOD

Method/guideline followed: Not indicated

Test type: E. coli DNA repair test

GLP compliant?: Not indicated

Year study performed: 1978

Test species: Escherichia coli W3110/polA+ (ATCC No. 27325); E. coli P3478/polA- (ATCC 

No. 25947); Animals: Sprague-Dawley adult male rats, 200-225 grams

Metabolic activation: With and without (tested separately with S-9 metabolic activation from 

rat livers)

Concentrations tested: 2, 10 and 20 μl/plate

Statistical methods used: One-sided t-test 

Test method/conditions remarks:

Section A. Repair Test Without Metabolic Activation:

Overnight broth cultures of E. coli (0.1 ml for the polA+ strain and 0.3 ml for the polA- strain 

were added to 2 ml of molten medium HA (0.75% agar) supplemented with 5 μg thymine per ml 

in a 16 x 125 mm screw cap tube. The tubes are mixed on a Vortex-Genie mixer and the 

contents poured over a base plate of medium HA. Plates are rotated to distribute the overlay agar 

which is allowed to harden. After the overlay has set, a sterile filter paper disc is placed in the 

center of the plate and saturated with the test chemical. Plates are incubated overnight at 37°C 

and the zones of inhibition are measured and compared for the polA+ and polA- strains. 

Liquid samples of the stock solution are tested at 20, 10 and 2 μl per plate.
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Plates are made for each control and each concentration of test agent. The following controls are 

included:

1. Bacteria only

2. Solvent alone

3. Known positive compound: MMS: 10 μl per plate

4. Negative control: Chloramphenicol, 30 μg/plate

Section B. Repair Test With Metabolic Activation

1. Non-specific induction of liver enzymes - Aroclor 1254, a polychlorinated biphenyl, is used 

for the non-specific induction of liver enzymes in rats. Adult male Sprague-Dawley rats, 

weighing 200-225 g, are given a single intraperitoneal injection of 500 mg/kg Aroclor 1254 in 

corn oil. On day 5 post injection, animals are sacrificed by CO2 asphyxiation and the livers are 

removed and the S-9 fraction prepared. Animals have free access to food until 12-15 hours 

before sacrifice; water is available at all times.

2. Preparation of S-9 fraction of the liver homogenate – All operations are performed aseptically 

at 0-4°C using sterile solutions and glassware. After surgical removal and weighing, livers are 

minced in 3 volumes 0.15 M KCl and homogenized in a Potter Elvejhem tissue grinder at 120-

150 rpm. The homogenate is centrifuged at 9000 x g for 20 min. and the supernatant decanted 

and saved and designated the S-9 fraction. The S-9 is divided into 1 ml amounts and stored 

frozen at -80°C until the day of use.

3. Preparation of S-9 mix – On the day of the assay, sufficient S-9 for that day’s use is thawed 

and mixed with cofactors for use in the assay. S-9 mix contains per ml: S-9 (generally 40 μl to 

100 μl), 8mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, and 100 mM 

sodium phosphate, pH 7.4. Reagents for the S-9 mix are weighed and prepared fresh daily with 

the exception of sodium phosphate which is made as a stock solution of 0.2 M. The entire S-9 

mix is filter sterilized before use through a 0.45 u Nalgene disposable filter unit. S-9 mix is kept 

at 0-4°C until use; unused portions are discarded at the end of the day.

4. Standardization of S-9 for use in the assay – Each bulk preparation of S-9 is assays for aryl 

hydrocarbon hydroxylase (AHH) activity; protein is determined according to the method of 

Lowry et al.

Prior to use in the assay, each S-9 preparation is titrated for bacterial mutagenesis in the 

Salmonella/microsomal assay of Ames et al. Each preparation is tested at a wide range of 

concentrations (10 to 300 μl S-9 per ml of S-9 mix) with 3MC, BP, and 6-AC to determine the 

optimum amount of S-9 for use in a general screening procedure. This is usually in the range of 

50 to 100 μl of S-9 per ml of S-9 mix. We have found that the optimum amount of used in the 

Salmonella/microsomal assay is also the best concentration of S-9 for the repair test so a single 

standardization is done for both.

5. The repair test with metabolic activation – The assay is the same as that described in Section 

A, except 0.5 ml of S-9 mix is added to the overlay agar immediately before it is mixed and 

poured. Plates are incubated and zones of inhibition are measured as noted above.
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Controls for the assay with metabolic activation are bacteria and bacteria plus S-9 mix. Sterility 

controls on the test agent are done as noted above. One-half ml of the S-9 mix, minus both test 

agent and bacteria, is added to 2 ml overlay agar and poured onto a minimal medium base plate 

and incubated for sterility also. 

Chloramphenicol at 30 μg/plate serves as the negative control. Diethylnitrosamine (DEN) at 10 

μl/plate serves as the positive control.

RESULTS

Before an agent is reported to be either active or inactive in the DNA Repair test with E. coli, the 

following criteria must be met:

All sterility controls, including test compound and S-9, must be negative for bacterial growth. S9 mix must not give a zone of inhibition. The bacterial control plates must show a complete 

lawn of growth. Known positive test agents must show a difference in zones of inhibition for 

strains polA+ and polA-. There must be no difference in the zone of inhibition with either strain 

for negative control compounds. Any test which does not meet these criteria must be repeated 

on a different day.

The tests reported upon below met each of the above mentioned criteria and the test agent was 

evaluated accordingly.

The difference in diameters of the zones of inhibition between the two strains (polA+ and polA-) 

is shown not to be statistically significant. The addition of an exogenous source of liver enzymes 

to activate the test compound resulted in no statistically significant difference between the 

diameters of the zones of inhibition. For this test, liver homogenates from adult male rats which 

had been pretreated with Aroclor 1254 for the non-specific induction of hepatic enzymes served 

as the activation system. This preparation was used at a final concentration of 100 μl of S-9 per 

ml of S-9 mix.

A one-sided t-test assuming unequal variants was performed on the data to determine statistical 

significance at either the 0.05 or the 0.01 level.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Not indicated

Results remarks: The integrity of the tester strains, polA+ and polA-

, is demonstrated by the 

preferential cell kill of a strain polA- which lacks excision repair, by MMS and by DEN, the 

positive control compounds for this test.

CONCLUSIONS

Under the conditions of this test, DETDA did not produce a statistically significant difference in 

its ability to produce cell death between repair deficient (polA-

) and repair competent (polA+) 

strains of E. coli.
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STUDY RELIABILITY

[2] Reliable with restrictions. Several details are missing from the study including purity and 

composition of the test substance, method/guideline followed, GLP compliance, cytotoxicity and 

analytical data to verify actual concentrations used in the assay.

REFERENCES

Schechtman, L., Parmar, A., and Fabrizio, D. 1978. Activity of DETA in a Test for Differential 

Inhibition of Repair Deficient and Repair Competent Strains of Escherichia coli: DNA Repair. 

Performing Laboratory: Microbiological Associates. Bethesda, MD. Sponsor: Ethyl 

Corporation, Baton Rouge, LA.
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Study 6: In vitro mammalian cell transformation assay (Schechtman et al., 1978)

Title: Activity of DETA in in vitro mammalian cell transformation assay

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Synonym:

Diethyltoluenediamine (DETDA); Purity not specified 

 

Remarks: Referred to as “DETA”, identified as “Diethyltoluenediamine”, brown liquid 

METHOD

Method/guideline followed: Protocol of Schechtman and Kouri

Test type: In vitro mammalian cell transformation assay

GLP compliant?: Not indicated

Year study performed: 1978

Test species: BALB/3T3 Clone A31 mouse cells

Metabolic activation: Without

Concentrations tested: 1:10000, 1:15000, 1:20000 and 1:30000 (dilutions of test agent in 

reaction mixture)

Statistical methods used: None specified

Test method/conditions remarks: In vitro mammalian cell transformation tests with 

BALB/3T3 Clone A31 mouse cells are performed according to the methods of Schechtman and 

Kouri. Target cells are treated in suspension for 2 hours at 37°C with different concentrations of 

test chemical in the presence or absence of exogenously supplied subcellular hepatic 

metabolizing activity (S-9), washed by centrifugation, and plated for analysis of the cytotoxic 

effects of treatment and the induction of phenotypic transformation.

Positive and negative controls are included in every experiment. Benzo(a)pyrene (BP) or Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) serve as positive control compounds in the 

presence or absence, respectively, of exogenous S-9. When appropriate, cell culture (growth) 

medium, acetone or dimethylsulfoxide (DMSO), the solvent vehicle, +/- S-9 serves as the 

negative control.

Exponentially growing BALB/3T3 C1. A31 cells were cultured in 75 cm2 cell culture flasks and 

collected by trypsinization. Cells were seeded in triplicate dishes per condition at densities of 

250 and 500 cells per 60 mm dish for determinations of the relative cytotoxic effects of each 
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treatment and 1 x 104 cells per 60 mm dish in 12 replicate dishes per condition for 

determinations of the phenotypic transformation effects of each treatment. All cells were seeded 

in 4 ml complete growth medium.

Twenty-four hours after the cells were seeded, twice concentrated working solutions of the test 

chemical or the positive or negative controls were added 1:1 (v/v) to the medium in the dish; 

cells were then incubated for 24 hours at 37°C. After the exposure period all treatment media 

were withdrawn and the target cells were washed once with HBSS supplemented with 5% fetal 

bovine serum. Thereafter, the cells were replenished with 5 ml complete growth medium. The 

medium was changed twice each week for the duration of the assay.

Approximately 9-11 days after the initiation of the experiment, the cytotoxicity assay plates were 

fixed with 100% methanol, stained with 10% aqueous Giemsa, and scored for the number of 

surviving colonies in order to determine the relative plating efficiencies. Approximately 30-40 

days after initiation of the experiment, foci in the transformation assay plates were also fixed, 

stained, and scored for the morphologically transformed phenotype designated as Type III foci.

The cytotoxicity results are expressed in terms of plating efficiencies, i.e. the percent of the 

number of surviving colonies per dish relative to the number of cells seeded per dish, according 

to the protocol of Schechtman and Kouri (Appendix A). The transformation results are 

expressed in terms of transformation frequencies, i.e. the number of morphologically 

transformed (Type III) foci per number of cells at risk to the test agent, which is dependent upon 

the plating efficiencies of each test condition, according to the protocol of Schechtman and Kouri 

(Appendix B).

A preliminary cytotoxicity test was conducted to determine the dose range to be employed in the 

transformation assay. Whenever possible, the approximately LD50 concentration of the test 

chemicals plus 1 dose above and 1 dose below the LD50 were those used in the transformation 

assay. Cell culture (growth) medium, acetone, or DMSO (the solvent vehicle for the test agents) 

served as the negative control; 3-MC or MNNG was used as the positive control.

APPENDIX A: Standard Protocol for the Determination of the Cytotoxic Effects of Test 

Chemicals to BALB/3T3 C1. A31 Mouse Cells in the Absence of Exogenous Metabolic 

Activation: Plate Assay

1. BALB 3T3 C1. A31 cells from sub-confluent stock cultures are seeded at 2 different cell 

densities (250 and 500 cells per 60 mm dish) in triplicate dishes per condition, in 4 ml growth 

medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum, and 

incubated at 37°C in a humidified atmosphere of 5% CO2 + air.

2. Twenty-four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle 

(negative control) are added 1:1 (v/v) to the seeding medium; cells are incubated for 24 hours at 

37°C.

3. Final concentrations of test chemical to be tested initially will cover a dose range from 30-0.1 

μg/ml in approximate half-log dilutions. However, if desirable, the highest dose to be tested will 

be determined by the limits of solubility of the test agent.
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4. After a 24 hr exposure period, the treatment medium is withdrawn; cells are washed once with 

Hanks’ balanced salt solutions (pH 7.0) and resupplied with 5 ml fresh growth medium.

5. The medium in all plates is replaced every 3-4 days for the duration of the assay.

6. The plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and scored 7-10 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded) and relative colony-forming efficiency (RCE) (i.e. number 

of colonies in chemically-treated dishes relative to the number of colonies in solvent-treated 

controls).

7. The approximate LD50 is determined from the relative survival data. The LD50 is that dose of 

test chemical which is lethal to 50% of the target cells.

APPENDIX B: Standard Protocol for the Morphological Transformation of BALB/3T3 C1. A31 

Mouse Cells in the Absence of Exogenous Metabolic Activation: Plate Assay

1. BALB 3T3 C1. A31 cells from sub-confluent stock cultures are seeded at 3 different cell 

densities (250 and 500 cells per 60 mm dish, in triplicate dishes per condition, for cytotoxicity 

tests; 1 x 104 cells per 60 mm dish, 12 dishes per condition, for transformation tests) in 4 ml 

growth medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum, and 

incubated at 37 C in a humidified atmosphere of 5% CO2 + air.

2. Twenty-four hours later, 2x concentrations of potential carcinogen (i.e. test chemical (3 

doses), or positive control chemical) or solvent vehicle (negative control) are added 1:1 (v/v) to 

the seeding medium; cells are incubated for 24 hr at 37°C. The doses of test chemical used shall 

be based upon the results obtained in a preliminary cytotoxicity assay, and shall include the 

approximate LD50, one dose above and one dose below the LD50. (The LD50 is that dose which 

is lethal to 50% of the target cells.) If an agent is non-toxic, then the highest dose that will be 

used will be a function of the limits of solubility of the test agent.

3. Treatment medium is withdrawn; cells are washed once with Hanks’ balanced salt solution 

(pH 7.0) and resupplied with 5 ml fresh growth medium.

4. The medium in all plates is replaced twice each week for the duration of the assay.

5. The cytotoxicity plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and 

scored 7-10 days after initiation of the experiment for relative plating efficiency (RPE) (i.e. 

number of surviving colonies per number of cells seeded).

6. Four to six weeks after initiation of the experiment, the transformation test plates are fixed, 

stained and scored for morphologically transformed foci displaying the phenotypic properties 

designated Type III. The cells comprising such foci are characterized by their loss of contactinhibition of cell growth and division, resulting in a randomly oriented piled-up array of cells. 

Cellular constituents of such transformed foci grow into colonies in semi-solid agar (whereas 
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142

non-transformed cells do not) and are tumorigenic in vivo. Data is expressed in terms of a 

morphological transformation frequency (TF). The TF data can be expressed in one of two 

ways: (a) if RPE data is available, then TF is expressed per number of cells at risk to the test 

agent, and (b) if RPE data is unavailable, the TF is expressed as a function of the number of 

dishes showing morphological transformants.

7. Representative morphologically transformed foci are isolated, grown to large cell populations 

(2-3 weeks) and confirmed for in vitro phenotypic transformation by growth in semi-solid agar. 

The semi-solid agar test takes 2-3 weeks.

RESULTS

In the preliminary study to determine the cytotoxic effects of compound DETDA on the 

BALB/3T3 C1. A31 cells, a dose response was not observed due to the extreme toxicity of 

DETDA to the target cells. The test agent was 100% cytotoxic at all of the concentrations tested 

although a few single cells were observed at the 1:1000 dilution; however, no colony formation 

was observed. Compound DETDA precipitated heavily at all of the concentrations tested.

In the additional preliminary study utilizing revised dilutions to determine the cytotoxic effects 

of DETDA on BALB/3T3 C1. A31 cells and to establish the approximate LD50 of the test 

compound to the target cells, DETDA was observed to be 100% cytotoxic at the 1:1000, 1:2000 

and 1:3000 dilutions. Although cell survival and colony formation was observed at the 1:5000 

dilution, the relative survival of the target cells at this concentration was judged to be too low to 

be used in the transformation assay.

In the cytotoxicity study of the compound DETDA which accompanied the transformation assay, 

a dose response was observed. Although DETDA was virtually 100% cytotoxic at the 1:10000 

dilution (0.69% relative survival), the % relative survival at the 1:15000, 1:20000, and 1:30000 

dilutions was found to be acceptable for the completion of the transformation assay (21.40%, 

30.0% and 42.8%, respectively). The approximate LD40 was found to be at the 1:30000 dilution 

of compound DETDA.

In the cell transformation test in the absence of exogenous metabolic activation, DETDA was 

ineffective in producing Type III foci at all of the concentrations tested. The negative control 

condition (medium control) exhibited no spontaneously transformed foci whereas, the positive 

control condition (MNNG) produced 4 morphologically transformed foci (transformation 

frequency of 6.0 x 10-4).

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 1:30000 (dilutions of test agent in reaction mixture) 

Results remarks: None specified
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CONCLUSIONS

Based on the results and discussion the following conclusions were made: (1) DETDA was 

found to be extremely cytotoxic to the BALB/3T3 C1. A31 cells. (2) DETDA precipitated in 

complete medium at all dilutions tested < 1:10000. (3) A dose response was observed only 

among dilutions tested ≥ 1:10000. (4) Under the conditions of the assay employed, test 

compound DETDA was incapable of inducing morphological transformants (type III foci) at any 

of the concentrations tested.

STUDY RELIABILITY

[2] Reliable with restrictions. Important details were missing from the study including test 

substance composition and purity, GLP compliance and statistical methods used.

REFERENCES

Schechtman, L., Beard, S., and Fabrizio, D. 1978. Activity of DETA in In Vitro Mammalian 

Cell Transformation Assay. Study No. T1365B. Performing Laboratory: Microbiological 

Associates., Bethesda, MD. Sponsor: Ethyl Corporation, Baton Rouge, LA.
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Study 7: In vitro mammalian cell transformation assay (Schechtman et al., 1979)

Title: Activity of T1365A in in vitro mammalian cell transformation assay in the presence of 

exogenous metabolic activation

TEST SUBSTANCE 

Identity: Diethyltolueneamine; Purity not specified

 

Remarks: Referred to as “DETA” and T1365A, identified as “Diethyltolueneamine”, brown 

liquid

METHOD

Method/guideline followed: Protocol of Schechtman and Kouri

Test type: In vitro mammalian cell transformation assay

GLP compliant?: Not indicated

Year study performed: 1979

Test species: BALB/3T3 C1.A31 cells

Metabolic activation: With

Concentrations tested: 1:1 x 104

, 1:1.5 x 104 and 1:2 x 104 (dilutions of test agent in reaction 

mixture)

Statistical methods used: None specified

Test method/conditions remarks: In vitro mammalian cell transformation tests with 

BALB/3T3 Clone A31 mouse cells are performed according to the methods of Schechtman and 

Kouri. Target cells are treated in suspension for 2 hours at 37°C with different concentrations of 

test chemical in the presence or absence of exogenously supplied subcellular hepatic 

metabolizing activity (S-9), washed by centrifugation, and plated for analysis of the cytotoxic 

effects of treatment and the induction of phenotypic transformation.

Positive and negative controls are included in every experiment. Benzo(a)pyrene (BP) or Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) serve as positive control compounds in the 

presence or absence, respectively, of exogenous S-9. When appropriate, cell culture (growth) 

medium, acetone or dimethylsulfoxide (DMSO), the solvent vehicle, +/- S-9 serves as the 

negative control.

Exponentially growing BALB/3T3 C1. A31 cells were cultured in 75 cm2 cell culture flasks and 

collected by trypsinization into 50 cc conical tubes for treatment in suspension at 27°C for 2 
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hours in a buffer pool (reaction mixture) containing an NADPH-generating system. The 

treatment conditions were as follows:

a. Cells + S-9 mix + BP, 12.5 μg/ml (positive control)

b. Cells + S-9 mix +/- solvent vehicle (negative control)

c. Cells + S-9 mix + test chemical (3 doses)

Prior to use, an S-9 preparation is pre-treated for metabolic capacity in terms of aryl hydrocarbon 

hydroxylate (AHH) activity by a modification of published procedures.

After the exposure period, the target cells were washed and resuspended in complete growth 

medium (basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum. Cells from 

each test condition were seeded at 250 cells per 60 mm dish in triplicate (cytotoxicity assay 

plates), and at 1 x 104 cells per 60 mm dish, 12 plates per condition (transformation assay plates) 

in 5 ml growth medium and incubated at 37°C. The medium was changed twice each week for 

the duration of the assay.

Approximately 7-10 days after initiation of the experiment, cells in the cytotoxicity assay plates 

were fixed with 100% methanol, stained with 10% aqueous Giemsa, and scored for the number 

of surviving colonies in order to determine the relative plating efficiencies (RPE) according to 

the protocol of Schectman and Kouri (Appendix A).

Approximately 4-6 weeks after initiation of the experiment, cells in the transformation assay

plates were fixed, stained and scored for the morphologically transformed phenotype designated 

as Type III foci, according to the protocol of Schechtman and Kouri (Appendix B).

The transformation results are expressed in terms of transformation frequencies (TF) i.e., the 

number of morphologically transformed (Type III) foci per number of cells at rick (CAR) to the 

test agent, which is dependent upon the plating efficiencies of each test condition.

A preliminary cytotoxicity study was conducted to determine the dose range to be employed in 

the transformation assay. Whenever possible, the approximate LD50 concentration of the test 

chemical plus one dose above and one dose below the LD50 were those used in the 

transformation assay.

APPENDIX A: Standard Protocol for the Determination of the Cytotoxic Effects of Test 

Chemicals to BALB/3T3 C1. A31 Mouse Cells in the Presence of Exogenous Metabolic 

Activation: Suspension Assay

1. Two-3 x 106 logarithmically growing BALB/3T3 C1. A31 cells from sub-confluent stock 

cultures are collected into 50 cc capacity conical centrifuge tubes (two tubes per treatment 

condition) and centrifuged at 1000 RPM for 10 min at 4°C. 

2. The supernatants are discarded and the cell pellets are resuspended in 4 ml reaction mixture 

consisting of the S-9 mix (containing 100 μl S-9 (approximately 40 mg protein/ml) and cofactor 

pool) +/- test chemical. The test conditions include:

a. Cells + S-9 mix + BP, 12.5 μg/ml (positive control)
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b. Cells + S-9 mix +/- solvent vehicle (negative control)

c. Cells + S-9 mix + test chemical

Final concentrations of the test chemical employed are those used in the accompanying 

mutation/transformation assay.

3. The tubes containing each reaction mixture are incubated for 2 hr at 37 C in a humidified 

atmosphere of 5% CO2 + air. The reaction is terminated by centrifugation at 1000 RPM for 10 

min at 4°C.

4. The cell supernatants are discarded; the cell pellets are resuspended for washing in Hanks’ 

balanced salt solution and then centrifuged at 1000 RPM for 10 min at 4 C.

5. The cell supernatants are discarded and the cell pellets are resuspended in growth medium 

(basal medium, Eagle) containing 10% heat-inactivated fetal bovine serum. A cell count is then 

taken on the cells pooled for each test condition.

6. Cells are seeded at 250 cells per 60 mm dish in triplicate dishes per condition, in 5 ml growth 

medium and incubated at 37°C.

7. The medium in all plates is replaced every 3-4 days for the duration of the assay.

8. The plates are fixed (methanol, 100%) stained (Giemsa, 10% aqueous) and scored 7-10 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded), and/or relative colony-forming efficiency (RCE) (i.e. 

number of colonies in chemically treated dishes relative to the number of colonies in solventtreated controls).

APPENDIX B: Standard Protocol for the Morphological Transformation of BALB/3T3 C1. A31 

Mouse Cells in the Presence of Exogenous Metabolic Activation: Suspension Assay

1. Two-3 x 106 logarithmically growing BALB/3T3 C1. A31 cells from sub-confluent stock 

cultures are collected into 50 cc capacity conical centrifuge tubes (two tubes per treatment 

condition) and centrifuged at 1000 RPM for 10 min at 4°C.

2. The supernatants are discarded and the cell pellets are resuspended in 4 ml reaction mixture 

consisting of the S-9 mix (containing 100 μl S-9 (approximately 40 mg protein/ml) and cofactor 

pool) +/- test chemical. The test conditions include:

a. Cells + S-9 mix + BP, 12.5 μg/ml (positive control)

b. Cells + S-9 mix +/- solvent vehicle (negative control)

c. Cells + S-9 mix + test chemical (3 doses)

The doses of test chemical used shall be based upon the results obtained in a preliminary 

cytotoxicity assay, and shall include, whenever possible, the approximately LD50, one dose 

above and one dose below the LD50. If an agent is non-toxic, then the highest dose that will be 

used will be a function of the limits of solubility of the test agent.
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3. The tubes containing each reaction mixture are incubated for 2hr at 37°C in a humidified 

atmosphere of 5% CO2 + air. The reaction is terminated by centrifugation at 100 RPM for 10 

min at 4°C.

4. The cell pellets are resuspended for washing in Hanks’ balanced salt solution and then 

centrifuged at 100 RPM for 10 min at 4°C.

5. The cell supernatants are discarded and the cell pellets are resuspended in growth medium 

(basal, Eagle) containing 10% heat-inactivated fetal bovine serum. A cell count is then taken on 

the cells pooled for each test condition.

6. Cells are seeded at 2 different cell densities (250 cells per 60 mm dish, in triplicate dishes per 

condition, for cytotoxicity tests; 1 x 104 cells per 60 mm dish, 12 dishes per condition, for 

transformation tests) in 5 ml growth medium and incubated at 37°C.

7. The medium in all plates is replaced twice a week for the duration of the assay.

8. The cytotoxicity plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and 

scored 7-10 days after initiation of the experiment for relative plating efficiency (RPE) (i.e. 

number of surviving colonies per number of cells seeded).

9. Four to six weeks after initiation of the experiment, the transformation test plates are fixed, 

stained and scored for morphologically transformed foci displaying the phenotypic properties 

designated Type III. The cells comprising such foci are characterized by their loss of contact 

inhibition of cell growth and division, resulting in a randomly oriented piled up array of cells.

10. Data is expressed in terms of morphological transformation frequency. The TF data can be 

expressed in one of two ways: (a) if (illegible) is vailable, then TF is expressed per number of 

cells at risk to the test agent, and (b) if RPE data is unavailable, then TF is expressed as a 

function of the number of dishes showing morphological transformants per condition.

APPENDIX C: Standard Protocol for the Determination of the Cytotoxic Effects of Test 

Chemicals to BALB 3T3 C1. A31 Mouse Cells in the Absence of Exogenous Metabolic 

Activation: Plate Assay

1. BALB/3T3 C1. A31 cells from sub-confluent stock cultures are seeded at 250 cells per 60 mm 

dish triplicate dishes per condition, in 4 ml growth medium (basal medium, Eagle) containing 

10% heat-inactivated fetal bovine serum, and incubated at 37°C in a humidified atmosphere of 

5% CO2 + air.

2. Twenty-four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle 

(negative control) are added 1:1 (v/v) to the seeding medium; cells are incubated for 24 hr at 

37°C.
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3. Final concentrations of test chemical to be tested initially will cover a dose range from 30-0.1 

μg/ml in approximate half-log dilutions. However, if desirable, the highest dose to be tested will 

be determined by the limits of solubility of the test agent.

4. After a 24 hour exposure period, the treatment medium is withdrawn; cells are washed once 

with Hanks’ balanced salt solution (pH 7.0) and resupplied with 5 ml fresh growth medium

5. The medium in all plates is replaced every 3-4 days for the duration of the assay.

6. The plates are fixed (methanol, 100%), stained (Giemsa, 10% aqueous) and scored 7-19 days 

after initiation of the experiment for relative plating efficiency (RPE) (i.e. number of surviving 

colonies per number of cells seeded), and/or relative colony-forming efficiency (RCE) (i.e. 

number of colonies in chemically-treated dishes relative to the number of colonies in solventtreated controls).

7. The approximate LD50 is determined from the relative survival data. The LD50 is that dose of 

the test chemical which is lethal to 50% of the target cells.

RESULTS

Under the conditions of the preliminary study (appendix C), compound T1365A was extremely 

cytotoxic to the target cells. The test agent was 100% cytotoxic to all of the concentrations 

tested although a few single cells were observed at the 1:1000 dilution; however, no colony 

formation was observed. Compound T1365A precipitated heavily at all of the concentrations 

tested.

In an additional preliminary study utilizing revised dilutions, T1365A was observed to be 100% 

cytotoxic at the 1:1 x 103, 1:2 x 103 and 1:3 x 103 dilutions. Although cell survival and colony 

formation was observed at the 1.5 x 103 dilution, the relative survival of the target cells at this 

concentration was judged to be too low to be used in the transformation assay.

These results necessitated an additional cytotoxicity assay which would serve to estimate the 

approximate LD50 of the test compound in the presence of exogenous metabolic activation. A 2 

hour suspension assay in the presence of S-9 activation was conducted to meet this necessity. 

Although quantitative results (i.e. colony formation, REP) were not established, a qualitative 

estimation of the relative toxicity of test agent to the garget cells expressed in terms of (%) 

relative survival after treatment could not be established. Under these conditions, compound 

T1365A was again observed to be extremely cytotoxic, even at a 1:1 x 104 dilution (Relative 

Survival ≤10%). A dramatic decrease in the level of toxicity was observed at dilutions ≥1:1.5 x 

104. The observed cellular response made it difficult to establish an LD50 for test agent T1365A. 

Therefore, the transformation assay (and its accompanying cytotoxicity assay) was performed 

using these same doses in order to bracket the LD50 range.

A quantitative cytotoxicity study accompanied the transformation assay. Under the conditions of 

this assay, an approximate LD50 was observed at the 1:1 x 104 dilution of test compound T1365A 

in the presence of exogenously supplied S-9. Little or no toxic effects were observed at the other 

doses of T1365A employed.
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In the transformation test in the presence of metabolic activation, the compound was ineffective 

in producing Type III foci at all of the doses tested. Treatment with BP (the positive control 

chemical) in the presence of exogenous S-9 induced 3 morphologically transformed (Type III) 

foci (transformation frequency of 1.6 x 10-4), whereas the negative control condition (S-9 + 

buffer) failed to induce spontaneous transformants.

The transformation frequencies at 1:1 x 104

, 1:1.5 x 104, and 1:2 x 104 were <0.55 x 104, <0.26 x 

104 and <0.23 x 104

, respectively.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 1:2 x 104 (dilutions of test agent in reaction mixture)

Results remarks: None specified

CONCLUSIONS

Based on the results and discussion the following conclusions were made. (1) T1365A was 

found to induce a cytotoxic response among BALB/3T3 C1. A31 cells both in the presence and 

absence of metabolic activation. (2) Over precipitation of T1365A during the treatment period 

was noted. Due to the limited solubility of test compound T1365A in vehicles compatible with 

mammalian cell culture systems, it was difficult to establish the exact concentration of that 

chemical in solution. (3) Under the conditions of the assay employed, the test chemical was 

ineffective in inducing morphological transformation (Type III foci) in the BALB/3T3 Clone 31 

cell system at all concentrations employed.

STUDY RELIABILITY

[3] Not reliable. Important details were missing from the study including test substance purity 

and composition, GLP compliance and statistical methods used. The test substance is identified 

as diethyltolueneamine, which differs from diethyltoluenediamine (the subject of required 

testing); diethyltolueneamine is not an acceptable analog for diethyltoluenediamine.

REFERENCES

Schechtman, L., Beard, S., and Sinsky, P. 1979. Activity of T1365A in In Vitro Mammalian 

Cell Transformation Assay in the Presence of Exogenous Metabolic Activation. Study No. 

T1365A. Performing Laboratory: Microbiological Associates, Bethesda, MD. Sponsor: Ethyl 

Corporation, Baton Rouge, LA.
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D-2. Carcinogenicity

Study 1: Two-year oncogenicity study in rats (Margitich and Ciofalo, 1992)

Title: Oncogenicity study – rat 24 month study

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Referred to as Diethyl 

Toluene Diamine (DETDA)

Remarks: Lot #6581-14; Clear yellow liquid; Analyzed composition of test substance 

throughout the study: 75.6-76.2% 2,4-Isomer; 20.0-20.2% 2,6-Isomer; 2.7-3.1% METDA 

(dialkylatedphenylenediamines); 0.2-0.3% TEMPDA (trialkylatedphenylenediamines); 126-181 

ppm 2,4-TDA (toluenediamine); 35-40 ppm 2,6-TDA (toluenediamine)

METHOD

Method/guideline followed: Protocol 474

Test type: Two-year oral oncogenicity study

GLP compliant?: Yes

Year study performed: 1988-1990

Test species: Sprague Dawley rats (Animals were 47 days old and had mean weights for males 

and females of 183.3 and 291.1 grams, respectively, at study initiation.)

Number of animals/sex/dose: 50

Route of administration: Oral (diet)

Vehicle: Certified commercial laboratory rat diet

Exposure duration: Daily, 7 days a week for 24 months (731 days)

Doses/concentrations tested: 0 (untreated control), 10, 35, 70 ppm

Statistical methods used: Raw data were collected and/or summarized and statistically 

evaluated using LABCAT modules designed by Innovative Programming Associates, Pnc., 303 

Wall Street, Princeton, NJ 08540. Evaluation of equality of means (body weight, food 

consumption, hematology and blood glucose) was made by the one way analysis of variance 

using the F distribution to assess significance. If significant differences among the means were 

indicated, Dunnett’s test was used to determine significant differences from control means. 

Statistical analyses (time-to-tumor) were evaluated using Systat (Version 4.1) by Systat Inc., 
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Evanston, IL. Estimates of the probabilities of survival for rats fed diets containing DETDA 

were calculated using - Kaplan and Meier statistics evaluated by SAS (Version 6.03), SAS 

Institute Inc., Gary, North Carolina. Statistical analysis of histopathological results were 

performed using the Fisher Exact Test and Cochran/Armitage Linear Trend Test.

Test method/conditions remarks: The commercial Diethyl Toluene Diamine (DETDA) 

product, Lot #6581-14, was incorporated into certified commercial laboratory rat diet and fed ad 

libitum to four groups of Sprague Dawley rats (50 animals/sex/group) seven days per week for 

731 days at dose levels of 0 (control), 10 (low), 35 (mid) or 70 (high) ppm. At the end of the 

feeding period, all surviving animals were sacrificed. During the study, animals were observed 

daily and weighed weekly. Food consumption was measured twice weekly when the diet was 

replenished. Blood was collected at approximately 6-month intervals for hematology exams 

(differential counts). Indirect ophthalmoscopy was performed on all rats prior to study initiation 

and at approximately 6, 12, 18 and 24 months. A complete necropsy was performed on all rats. 

Approximately 40 tissues were examined histologically from all rats in the control and high dose 

groups and all rats that died or were sacrificed during the study. The adrenal glands, eyes, liver, 

pancreas, pituitary and thyroid glands were evaluated in the mid and low dose groups. The 

mammary glands and any tissue mass suspected of being a mammary gland tumor were 

evaluated in the low and mid dose females. 

RESULTS

Endpoint value(s): LOAEL (hepatotoxicity) = 10 ppm (based on hepatic proliferative lesions 

and increased incidence and severity of degeneration changes in the liver of males); NOAEL 

(systemic toxicity) = Not established; LOAEL (pancreatic toxicity, males) = 70 ppm (based on 

pancreatic acinar atrophy accompanied by interstitial fibrosis and fatty infiltration); LOAEL 

(pancreatic toxicity, females) > 70 ppm (based on no adverse effects at the highest dose tested); 

NOAEL (pancreatic toxicity, males) = 35 ppm; NOAEL (pancreatic toxicity, females) = 70 ppm; 

LOAEL (thyroid effects) = 70 ppm (based on thyroid hyperplasia and hypertrophy); NOAEL 

(thyroid effects) = 35 ppm; LOAEL (liver and thyroid tumors) = 70 ppm (based on increased 

hepatocellular carcinomas and follicular cell adenomas in males, and increased hepatocellular 

adenomas in females); NOAEL (liver and thyroid tumors) = 35 ppm; NOAEL (mammary 

tumors) ≥ 10 ppm (based on questionable significance of mammary tumors in the mid and high 

dose females)

Results remarks: The majority of clinical signs observed were related to aging. These signs 

were not considered related to the administration of DETDA as they were distributed across the 

four groups in a non-dose-related manner. Ocular opacity was noted in a few high dose males. 

This change is considered the only clinical sign, which may have been related to the 

administration of the test article.

Mean body weight of the male high dose group was statistically decreased from the control

male body weight beginning on week 43 and continuing throughout the majority of the

study. Control and mid dose mean body weights were statistically comparable throughout the 

study except on one occasion. However, a tendency for lower mean body weight in the mid dose 

males was seen beginning at about month 15 and continuing to the conclusion of the study. No 
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effect, statistical or otherwise, was found on the body weight of low dose males compared to 

control males.

The high dose female mean body weight was statistically comparable to the control female body 

weight except during four weeks of the study (weeks 80 - 83). During these 4 weeks, mean body 

weight of the high dose females was statistically lower than the female control mean. A 

tendency for a lower body weight in the high dose females compared to the control mean was 

seen in the last 3 months of the study. No statistical differences between the control and middose female body weight were found. A tendency for a lower body weight in the mid dose was 

seen during the last four weeks of the study. The low dose female body weight was not affected.

Group mean food consumption was not affected by the administration of DETDA. Only 

sporadic, non-dose-dependent statistical differences in food consumption were detected between 

control and treated animals of either sex. However, a few male high dose DETDA rats 

consistently exhibited a marked increase in daily food consumption.

No ocular lesions related to the administration of DETDA were detected on indirect 

ophthalmoscopy with the exception of bilateral cataracts in several high dose male rats. A 

correlation between animals affected with ocular cataracts and blood glucose levels greater than 

300 mg/dL was observed at the 18-month blood collection. No treatment related effect on white 

blood cell differential count or red cell morphology was found at any time point.

Survival was comparable between treated and control animals of each sex except in the low dose 

male rats. Survival in this group was significantly greater than the control males. Percent 

survival was maintained above 50% until week 97, 104, 96 and 92 for males in the control, low, 

mid- and high dose groups, respectively. In females, 50% survival was maintained up to week 

103, 102, 101 and 98 for the control, low, mid and high dose groups, respectively. Early deaths 

or moribund sacrifices occurred during the study. Lesions considered as the cause of death or 

moribund sacrifice included pituitary tumors, other large and/or malignant neoplasms, severe 

chronic renal disease and severe lesions of the hock/foot (suppurative and ulcerative dermatitis). 

Rats in this study reached high body weights early in the course of the study. This factor may 

have contributed to the development of certain of the changes leading to the early deaths or 

moribund sacrifices.

The majority of gross macroscopic observations at terminal necropsy were associated with aging 

and not considered manifestations of toxicity due to treatment. Certain gross changes in the liver 

may have been related to the test article.

Histopathology results showed a statistically increased incidence in comparison to the control 

animals of hepatocellular carcinomas and proliferative lesions in the liver of the high dose males, 

hepatocellular adenomas and proliferative lesions in the liver of the high dose females, thyroid 

follicular cell adenomas and follicular cell hypertrophy in high dose males and fibroadenomas of 

the mammary glands of mid and high dose females. Hepatic proliferative lesions (basophilic 

foci and eosinophilic foci) were significantly increased in all three male dose groups. All three 

male dose groups had an increased incidence and severity of degeneration changes in the liver 

(focal and multifocal cystic degeneration and multifocal necrosis). Basophilic foci in the liver of 
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high dose females were increased relative to controls. Eosinophilic foci in the liver of low dose, 

but not mid- or high dose, females were increased compared to control females. Pancreatic 

acinar atrophy accompanied by interstitial fibrosis and fatty infiltration (fatty atrophy) was 

present in the high dose male rats. Bilateral cataracts were detected in six high dose male rats. 

The presence of moderate to severe keratitis in half these animals complicates the interpretation 

of the significance of the cataracts. However, based on the early detection of cataracts by 

indirect ophthalmoscopy coupled with elevated blood glucose levels, it appears likely these 

changes were related to administration of test article. Renal cysts and focal areas of tubular 

hyperplasia were increased in high dose males compared to the control male rats. The relevance 

of treatment to this effect is equivocal, since some degree of age associated chronic nephropathy 

was present in nearly all of the kidneys examined and cysts and tubular hyperplasia are 

components of chronic nephropathy. No treatment-related neoplastic or nonneoplastic changes 

were noted in any of the other tissues examined. A variety of spontaneous neoplasms and 

incidental lesions were present in both the control and DETDA treated rats.

With respect to tumorigenicity, hepatocellular carcinomas were increased in the male high dose 

group. The incidence of a benign tumor of the thyroid, follicular cell adenoma, was also 

increased in high dose males. Hepatocellular adenoma, a benign tumor of the liver, was 

increased in high dose female rats. Mammary gland fibroadenoma, a benign tumor, were 

increased in mid- and high dose females. The effect on mammary gland tumors is the only 

instance where the female rats appeared affected at a lower level than the male. Since the 

incidence of malignant mammary gland tumors (adenocarcinomas) was high in the control 

females, the relevance of the increase in fibroadenomas is unknown. A no effect level for liver 

and thyroid tumors was 35 ppm. The questionable significance of the mammary tumors in the 

mid and high dose females complicates determination of a no effect level in this organ. At a 

minimum, the no effect level in this organ is 10 ppm and may be higher.

The results of this study should be evaluated in conjunction with the earlier DETDA studies 

conducted in the same laboratory. The 90-day subchronic and 28-day progression/reversibility 

studies identified the pancreas as the target organ in the rat. Male rats were more severely 

affected and at an earlier time and lower dose than females. Histologically, pancreatic acinar 

cells were affected first; islet cell involvement followed afterward in a time and dose dependent 

manner. Pathology in other organs occurred after or in conjunction with islet cell involvement 

and appeared secondary to the metabolic changes induced by the pancreatic toxicity. Secondary 

effects included ocular cataracts in animals with histologic evidence of islet cell toxicity. It was 

postulated these cataracts were associated with and related to hyperglycemia and thus the 

pancreatic toxicity. Histologic evidence of liver or thyroid toxicity was not seen.

The greater DETDA sensitivity of male, compared to female, rats was also observed in the twoyear study. This response holds true for both toxicity and tumorigenicity. However, effects on 

the liver and thyroid, not seen in the subchronic studies, were detected in the two-year study. 

Based on the absence of these effects in shorter term studies, liver and thyroid effects appear 

related to chronic, long term exposure.
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CONCLUSIONS

In the two-year study, histologic evidence of pancreatic toxicity was restricted to the pancreatic 

acinar cells of high dose male rats. A functional aberration was suggested in individual rats by 

the hyperglycemia, increased food consumption and ocular cataracts observed in a few high dose 

male rats. No histologic or suggestive evidence of pancreatic toxicity was seen in the mid and 

low male dose groups or in females at any dose level. Given these results, the no effect level for 

pancreatic toxicity in the male rat for two years was 35 ppm; for females the no effect level was 

70ppm.

Unlike pancreatic toxicity, hepatic proliferative and degenerative changes were found in all male 

dose groups. Only proliferative lesions were found in the females. A clear no effect level was 

not achieved. However, hepatotoxicity would appear related to long term chronic exposure as 

this effect was not observed in studies less than two years in length, e.g., the no effect level for 

hepatotoxicity was 320 ppm (highest dietary concentration tested) DETDA in a 90-day dietary 

subchronic study in Sprague-Dawley rats of [see review of this study (Naismith and Matthews 

1987) in a separate robust summary within this document].

Evidence for thyroid hyperplasia or hypertrophy was found in high dose males and females. 

Lower dose levels were not affected. Like hepatotoxicity, thyroid effects were not seen in 

subchronic studies and are an effect of chronic long-term exposure. A no effect level for both 

males and females in this study is 35 ppm.

Evidence of tumorigenicity was observed in the livers and thyroids of males dosed at 70 ppm and 

in the livers of females dosed at 70 ppm. Evidence of mammary tumors in females dosed at 35 

or 70 ppm is of questionable significance; therefore, at a minimum, the no effect level in this 

organ is 10 ppm and may be higher.

STUDY RELIABILITY

[1] Reliable without restrictions. 

REFERENCE

Margitich, D. and Ciofalo, V. 1992. Oncogenicity Study - Rat. 24 Month study. Study No. PH 

474-ET-001-88. Diethyl Toluene Diamine (DETDA). Lot# 6581-14. Performing Laboratory: 

Pharmakon Research International Inc. Waverly, PA. Sponsor: Ethyl Corporation, Baton Rouge, 

LA.
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Study 2: Lifetime oral carcinogenicity (Steinhoff, 1984)

Title: Diethyltolamine carcinogenicity study with oral administration to rats

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA)

Remarks: Referred to as “diethyltolamine”, but identified as diethyltoluenediamine. Batch 74 

of the test substance was used for the preliminary experiments and for the first 11.5 months of 

the feeding study and Batch 94 was used for the remainder of the treatment in the feeding study 

and for the entire gavage study. Analysis of Batch 74 revealed a 2,4-DETDA content of 66.8% 

and a 2,6-DETDA content of 28.9%; Viscous brown liquid 

METHOD

Method/guideline followed: Not indicated

Test type: Lifetime oral carcinogenicity study

GLP compliant?: Not indicated

Year study performed: 1978-1982

Test species: Wistar-W74 rats; Animals were 6 weeks old at test initiation

Number of animals/sex/dose: Gavage study - 20; Diet study – 50, plus 5/sex/group for interim 

sacrifices after 1 and 2 years

Route of administration: Oral (gavage or diet)

Vehicle: Gavage study – Arachis oil; Diet study - Feed

Exposure duration: Gavage study – 2 years; Diet study - Lifetime

Doses/concentrations tested: Gavage study – 0 (untreated control), 0 (vehicle control), 4 or 12 

mg/kg-bw/day; Diet study – 0 (control), 80 or 240 ppm

Statistical methods used: 

DETDA in the feed:

“Dead” and “moribund sacrificed” are treated as equivalent for the calculation of survival rates, 

i.e. “moribund sacrificed” is not regarded as censorable data. The rates are calculated according 

to KAPLAN-MEIER are therefore relative frequencies. The outcome of the three dose groups 

per gender are compared to each other by means of generalized SAVAGE test.
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The number of animals with the histopathological tumor diagnoses are ordered according to 

topographical details and then portrayed separately according to gender. The animals are also 

arranged according to the number of locations with a positive tumor finding and according to the 

dignity of the findings.

Data is assembled for tumors that appear in a minimum of four animals of one gender within at 

least one treatment group as well as for benign, malignant and (positively identified) tumors. 

The cumulative numbers of animals with a particular tumor are tabulated at weekly intervals.

The possibility of a relationship between the tumor findings and the dosage is tested on the basis 

of recommendations by PETO et al. The data as such still do not permit a causal relationship 

between a diagnosed tumor and the death of an animal to be distinguished from a coincidental 

one, however.

This information would be required for the calculation of the “death by tumor” rates (death rate 

method) or the tumor prevalence rates. In place of this, the hypothesis is tested – formally in the 

sense of the “prevalence method” – that the probability of positive tumor findings in animals 

which die or are sacrificed in a moribund state within the same time span is the same for all 3 

dose groups for each gender. This is set against the one-sided alternative that this probability 

increases with increasing dosage of diethyltolamine. Weighting factors applied to the testing of 

these hypotheses are 0, 1 and 2 (increasing with dose).

Such tests are not carried out because the relevant paired comparisons (e.g. against controls) are 

not available for a given multiple niveau and it is not the objective of the study to determine the 

dose above which a clear-cut carcinogenic effect is seen. The results of both trend tests (one per 

gender and in each case not significant) also fail to provide a basis for going beyond a purely 

descriptive analysis of the individual tumor diagnoses.

The evaluation of the data is carried out by means of an ad hoc programme (cumulative number 

of animals with tumors seen against a time scale), the routine methods of the programme 

packages SAS (enumerating the tumor diagnoses) and BMDP (survival rates) and a programmed 

developed previously in the Department of Biometrics, EP-FE (Prevalence rates by Dr. Dycka).

Administration of DETDA by gavage:

“Dead” and “moribund sacrified” are treated as equivalent for the calculation of survival rates, 

i.e. “moribund sacrified” is not regarded as censorable data. The rates are calculated according 

to KAPLAN-MEIER are therefore relative frequencies. The outcome of the three dose groups 

per gender are compared to each other by means of generalized SAVAGE test.

The number of animals with the histopathological tumor diagnoses are ordered according to 

topographical details and then portrayed separately according to gender. The animals are also 

arranged according to the number of locations with a positive tumor finding and according to the 

dignity of the findings.
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Data is assembled for tumors that appear in a minimum of four animals of one gender within at 

least one treatment group as well as for benign, malignant and (positively identified) tumors. 

The cumulative numbers of animals with a particular tumor are tabulated at weekly intervals.

The possibility of a relationship between the tumor findings and the dosage is tested on the basis 

of recommendations by PETO et al. The data as such still do not permit a causal relationship 

between a diagnosed tumor and the death of an animal to be distinguished from a coincidental 

one, however.

This information would be required for the calculation of the “death by tumor” rates (death rate 

method) or the tumor prevalence rates. Moreover, there are no known methods that are relevant 

to the particular problems associated with this type of study and which are able to present linear 

contrasts in the manner of analysis of variance. It is, however, necessary to consider whether 

untreated and oil-treated controls can be combined for the assessment and compared to the two 

groups receiving substance. This in turn determines whether tests for inhomogeneity or trend 

should be made.

The test is therefore carried out in two stages: firstly, the hypothesis is tested – formally in the 

sense of the “prevalence method” – that the probability of positive tumor findings in animals, 

which die or are sacrificed in a moribund state within the same time span, is the same in both 

control groups or, as the alternative hypothesis, that it is different. On the basis of the results of 

this test, the two control groups are combined and used to test the hypothesis described above 

and the one-sided alternative hypothesis, and the probability of tumor occurrences increases with 

dose. Weighting factors applied to the testing of these hypotheses are 0, 1 and 2 (increasing with 

dose). 

The generalized evaluation by the prevalence method leads to only 2 time interval classes in the 

female animals when the time course is taken into consideration. Since only one animal dies in 

the first time interval the analysis is ended at this point.

The evaluation of the data is carried out by means of the routine methods of the programme 

packages SAS (description of the tumor findings) and BMDP (survival rates) and a programme 

developed previously in the Department of Biometrics, EP-FE (Prevalence rates by Dr. Dycka).

Test method/conditions remarks: Preliminary acute and 90-day studies were conducted. The 

acute toxicity of DETDA was determined using Batch 74 (determination of the LD50). The 

substance was mixed with arachis oil and administered by gavage on a single occasion to groups 

of 10 female Sprague-Dawley rats aged about 8 months (breeder: Mus-Rattus AG). The 

administration volume was 5 ml/kg. The observation period lasted three weeks. All the animals 

were autopsied. The LD50 was calculated by means of probit analysis.

The 90-day study was based on four groups of 10 Wistar rats (5/sex) aged about 8 months. In 

this preliminary study the DETDA (Batch 74) was mixed in with the powdered feed. In 

concentrations of 0, 100, 300 and 900 mg/kg feed (ppm). The effect of the DETDA was 

assessed on the basis of the rats’ appearance and behavior, their body weights and their feed 

consumption and on the results of the autopsies performed at the end of the study.
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Carcinogenicity study: In one experimental group, the DETDA was mixed in with the powdered 

feed in initial concentrations of 0, 80 and 240 ppm; in a second group the DETDA was 

administered by gavage in initial doses of 0, 4, and 12 mg/kg body weight five times a week. In 

the feeding study 50 male and 50 female rats were used for each group (with additional 5 

rats/sex/group for interim sacrifices after 1 and 2 years), while in the group with administration 

by gavage each group comprised 20 male and 20 female rats. The experiments were performed 

as lifetime studies, i.e. all the rats died a natural death or were sacrificed in a clearly moribund 

state. In the feeding treatment the DETDA was given until the end of the study; in the gavage 

administration the substance was given for 2 years (5 days/week) and the rats then continued to 

be observed until they died naturally.

The high dose in both dietary and gavage administration was clearly toxic, especially in male 

rats. Consequently, the low and high dietary doses were halved from study 456 onward. The 

gavage doses were also reduced by 50% from day 127 until day 256 when the doses were halved 

again. The final doses administered via gavage were 1 or 3 mg/kg 5 days per week. 

RESULTS

Endpoint value(s): No evidence of carcinogenicity.

Results remarks: After a single oral administration in arachis oil to female Sprague-Dawley rats 

DETDA produced non-specific symptoms of intoxication, manifested by general inertia with the 

animals assuming the prone position and lacrimating. No pathological findings were obtained at 

the autopsies other than bloodiness of the lungs. The LD50 (confidence interval p ≤ 0.05) was 

598 (568-653) mg/kg body weight.

As in the acute toxicity study, the intoxication symptoms in the 90-day study proved to be 

uncharacteristic. A striking feature was the poor general condition of the rats receiving the 

highest concentration of DETDA (900 ppm in the feed). This applied to the male rats in 

particular. At the lower concentrations (300 and 100 ppm in the feed) no appreciable differences 

from the controls were found in either appearance or behavior. At the highest concentration (900 

ppm) the animals suffered a distinct loss of weight, a phenomenon that again applied most 

particularly to the males. At the lower concentrations (300 and 100 ppm) no appreciable 

differences from the control animals were found in this respect. During the 13-week study only 

one male rat receiving the highest concentration (900 ppm) died, this incident occurring in the 

12th week, without abnormal findings.

The food consumption calculated per kg body weight differed clearly from that of the control 

groups only in the group on the highest concentration of DETDA in the feed (900 ppm), an 

increase being found towards the end of the study both in the males and in the females compared 

with the controls and the animals receiving the lower concentrations. 

The autopsies of the animals in the highest concentration group (900 ppm) performed at the end 

of the study did not yield any particular findings.
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Results for carcinogenicity study: Although clearly toxic, the nature of toxicity was not 

determined either by clinical observation or on histopathology. Rats treated with the test article 

via the diet or gavage demonstrated reduced body weights, poor general condition, increased 

food and water consumption, and shorter survival times. Males at the upper doses were more 

severely affected than females. 

No evidence of carcinogenicity was found. When results by the two methods of administration 

were considered together, a dose-dependent increase in the number of uterine tumors were 

counterbalanced by a more marked reduction in testicular tumors. Tumors of mammary gland 

were not dose-dependently increased. Tumors in the adrenal gland were reduced at the high 

dose. Two malignant kidney tumors were found in the low dose group, two in the high dose 

group and two in the controls. Three benign kidney tumors were found at the high dose.

CONCLUSIONS

The report concluded that no evidence of carcinogenicity was found despite the fact that the test 

animals were treated with the maximum tolerated to toxic doses.

STUDY RELIABILITY

[2] Reliable with restrictions. Test method/guideline and GLP compliance was not specified

REFERENCE

Steinhoff, D. 1984. Draft Report. Diethyltolamine Carcinogenicity Study with Oral 

Administration to Rats. Research Report. Performing Laboratory: Bayer AG Institute of 

Toxicology. Sponsor: Bayer AG, Germany.
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Study 3: Lifetime subcutaneous carcinogenicity (Steinhoff, 1972)

Title: Carcinogenicity Study with the Isomer Compound Diethyltolamine Technical 

Experimental Product DD 1443 Batch 1 in Subcutaneous Doses to Rats

TEST SUBSTANCE 

Identity: Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Synonym: 

Diethyltolenediamine (DETDA)

Remarks: Referred to as Diethyltolamine Technical Experimental Product DD 1443 batch 1; 

Content of 72.8% 3,5-diethyltoluene-2,4-diamine and 26.2% 3,5-diethyltoluene-2,6-diamine (the 

remainder consists of unknown compounds)

METHOD

Method/guideline followed: Not indicated

Test type: Lifetime subcutaneous carcinogenicity study

GLP compliant?: Not indicated

Year study performed: Not indicated

Test species: Wistar rats (Winkelmann W 64 and GfVz-Hannover)

Number of animals/sex/dose: Test animals: 10 females, 20/sex; Control animals: 10 females, 

25/sex

Route of administration: Subcutaneous injection

Vehicle: Physiological saline

Exposure duration: 510 days

Doses/concentrations tested: 1.6 and 3.15 g/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: The LD50 was determined after single subcutaneous doses to 

female specifically bred Winkelmann Wistar rats (W 64), weighing approximately 140 g. These 

were undiluted injections of the preparation. The LD50 determined was 368 (338-400) mg/kg (p 

= 0.05). The surviving rats showed a continuous degeneration of general condition during the 

four-week observation period.
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In the main study, one group of Wistar rats (20 males and 20 females of Winkelmann strain) was 

injected subcutaneously over a time period of 510 days with up to a total of 1.6 g DETDA/kg 

bw. A second group of rats served as the controls (25 males and 25 females), and were injected 

with physiological saline. A third group of 10 female Wistar rats of GfVz-Hannover strain, 

which appeared less susceptible to DETDA toxicity was injected with approximately double the 

amount or a total of 3.15 g DETDA/kg bw. Animals were not subjected to a scheduled sacrifice. 

Rather, they were allowed to survive over their natural lifespan. The dose used in the study was 

based on the LD50. The LD50 via subcutaneously injection was determined to be 368 mg/kg, and 

doses lower than this were selected for the carcinogenicity study.

RESULTS

Endpoint value(s): No evidence of carcinogenicity.

Results remarks: The average survival time of the 20 male Winkelmann rats treated with 

Diethyltolamine to a total dosage of 1.6 g/kg was 490 ± 349 experimental days. Of these 20 

male rats, 4 died with one malignant tumor each. These included a spindle-cell sarcoma of the 

larynx, a subcutaneous fibrosarcoma of the neck, a fibrosarcoma of the salivary gland, and a 

subcutaneous fibrosarcoma in the cheek. Benign tumors developed: a cystic papilloma and two 

subcutaneous fibromas.

The average survival time of the 20 female Winkelmann treated rats with Diethyltolamine 541 ± 

262 experimental days. Of these 20 female rats, 2 died with one malignant tumor each. One 

developed a uterine adenosarcinoma; the other developed a keratinized flat epithelial carcinoma 

of the breast. The benign tumors included 9 fibroadenomas of the breast, 10 uterine polyps, and 

1 uterine myoma.

The 25 male rats in the control group had an average survival time of 834 ± 229 days. Four of 

them died with one malignant tumor each. These four tumors were: a skin carcinoma, a 

subcutaneous sarcoma of the head, an adenocarcinoma of the lung, and a polymorphous-cells 

carcinoma of the brain. Besides these, a benign tumor of the thymus developed.

The average survival time of the 25 female rats of the control group was 888 ± 177 days. Nine 

of these rats died of malignant tumors, three of which were double tumors. These included: a 

large-celled carcinoma of the lung, a carcinomatous deteriorated fibrolipoma of the breast, two 

adenocarcinomas of the breast, an ovarian carcinoma, a sarcoma of the thymus, three uterine 

adenocarcinomas, one case of myelogenous leukemia, a keratinized flat epithelial carcinoma of 

the ear, and an adenocarcinoma of the thyroid gland. In addition, 14 benign tumors were 

observed, namely fibroadenomas of the breast.

The following results were observed with the 10 GfVz-Hannover female Wistar rats used: the 

average survival time was 568 ± 357 days. One of these rats died with two fibrosarcomas in the 

breast area. Besides these, 6 benign tumors developed – 4 fibroadenomas of the breast, one 

uterine polyp, and a lipoma on the thorax.
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When the last of the 10 experimental rats of this group had died, 6 of 10 rats in the corresponding 

control group had died. Until this time, there had only been 5 benign tumors observed in the 

control group: 3 fibroadenomas of the breast and 2 uterine polyps.

CONCLUSIONS

The isomer compound Diethyltolamine technical Experimental Product DD 1443 batch 1 

showed a clear toxicity after single subcutaneous doses to the rats (LD50 368 mg/kg), which 

showed itself primarily in a continuous degeneration of general condition. Consequently, no 

high doses could be given in the Carcinogenesis Study (total dosage: 1.6 g/kg). In spite of this 

only moderately high total dosage, the survival time in the experimental group was clearly 

decreased in comparison to the control group (an average of 516 days compared to 861 days).

All together, in the experimental group treated with technical isomer compound Diethyltolamine, 

among 40 rats (20 males, 20 females), there occurred 6 malignant tumors (15%), and 23 benign 

tumors (57%). In the control group treated with physiological NaCl solution, 16 malignant 

(32%) and 15 benign tumors (30%) were observed among the 50 rats (25 males, 25 females).

The additional 10 female GfVZ Wistar rats proved to be somewhat more resistant to 

Diethyltolamine. As a result, they received a total dosage of the preparation injection which was 

approximately double (3.15 g/kg). 

The survival time of these rats was also clearly reduced when compared to the corresponding rats 

treated with physiological NaCl solution. When the last experimental rat died, 4 of the animals 

in the control group were still living. Until this time, 2 malignant and 6 benign tumors had 

occurred in the experimental group: only 5 benign tumors had developed in the control group.

All together, then, among the 50 rats treated with technical Diethyltolamine, there developed 8 

malignant tumors (16%) and 29 benign tumors (58%); among the 60 rats treated with 

physiological NaCl solution, 16 malignant tumors (27%) and 20 benign tumors (33%) were 

observed.

There is, then, no indication from this study that technical isomer compound Diethyltolamine 

Experimental Product DD 1443 batch 1 has a carcinogenic effect after subcutaneous doses to 

rats. This conclusion is also reinforced by the fact that the malignant tumors which occurred in 

the experimental group showed quite varied localization, as was the case in the control group. 

The benign tumors in the experimental group were primarily of the same type frequently 

occurring in the untreated rat breeds used.

The high percentage of malignant and benign tumors in the control group can be explained by: 1) 

The special tendency of the breeds used toward tumor formation, and 2) the long survival times. 

It is also true that these animals were exposed to overall environmental conditions, and that, as is 

the case with man, cancer is primarily a disease of old age. The number of malignant tumors in 

the rats treated with Diethyltolamine – almost half the number of the control group – can be 

explained by the clearly reduced survival time.
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STUDY RELIABILITY

[2] Reliable with restrictions. Several details were missing from the study including test 

method/guideline followed, GLP compliance and statistical methods used.

REFERENCE

Steinhoff, D. 1972. Final Report on the Carcinogenesis Study with the Isomer Compound 

Diethyltolamine Technical Experimental Product DD 1443 Batch 1 in Subcutaneous Doses to 

Rats. Report No. 3709. Performing Laboratory: Bayer AG Toxicological Institute. Sponsor: 

Bayer AG, Germany.
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D-3. Skin Irritation

Study 1: Dermal irritation study in rabbits (Heenehan et al., 1977)

Title: Primary Dermal Irritation Study in Rabbits

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Primary Dermal Irritation -- Rabbit

GLP compliant?: Not indicated

Year study performed: 1977

Test species: New Zealand White albino rabbits

Number of animals/sex/dose: 6, sex not indicated

Route of administration: Dermal

Vehicle: None

Doses/concentrations tested: 0.5 mL

Statistical methods used: None

Test method/conditions remarks: The clipped skin of six New Zealand White albino rabbits 

was treated with 0.5 ml of DETDA at two sites: one abraded and one intact. Application sites 

were covered with an impervious bandage. After 24 hrs, the bandage was removed. Observations 

for dermal irritation or systemic toxicity were recorded at 24 and 72 hours after application. 

RESULTS

Endpoint value(s): Slightly irritating. The primary dermal irritation index based on erythema, 

edema and eschar formation was 1.7.
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Results remarks: None specified

CONCLUSIONS

The test substance was slightly irritating to rabbit skin.

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the study summary included purity and 

composition of the test substance, test method/guideline followed, observed effects and 

interpretation of irritation scores. 

REFERENCE

Heenehan, P., Braun, W., and Rinehart, W. 1977. Primary Dermal Irritation Study in Rabbits. 

Compound: Diethylated Toluenediamine. Project No. 4451-77. Performing Laboratory: 

Bio/dynamics Inc. Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in 

Albemarle Corporation. 2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR 

#335194.
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D-4. Eye Irritation

Study 1: Eye irritation study in rabbits (Heenehan et al., 1977)

Title: Rabbit Eye Irritation Study

TEST SUBSTANCE 

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Primary Eye Irritation -- Rabbit

GLP compliant?: Not indicated

Year study performed: 1977

Test species: New Zealand White rabbits

Number of animals/sex/dose: 6, sex not indicated

Route of administration: Ocular

Vehicle: None

Doses/concentrations tested: 0.1 mL

Statistical methods used: None specified

Test method/conditions remarks: One eye of each of six New Zealand White rabbits was 

treated with 0.1 mL DETDA. Observations were recorded for 14 days. 

RESULTS

Corneal opacity and ulceration were noted in all treated eyes. Iritis was noted in 3 of 6 eyes, 

persisting in one eye through Day 2. All six eyes scored positively for conjunctival redness. One 

eye was free of all signs of irritation on Day 3, three eyes on Day 4 and 2 eyes on Day 10. 
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Endpoint value(s): Irritating. A mean Draize Score of 27 was determined.

Results remarks: None specified

CONCLUSIONS

The test substance was irritating to rabbit eyes.

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the study summary included purity and 

composition of the test substance, test method/guideline followed and interpretation of irritation 

scores. 

REFERENCES

Heenehan, P., Braun, W., and Rinehart, W. 1977. Rabbit Eye Irritation Study. Compound: 

Diethylated Toluenediamine. Project No. 4450-77. Perfromeing Laboratory: Bio/dynamics Inc. 

Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle Corporation. 

2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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D-5. Skin Sensitization

Study 1: Dermal sensitization study in guinea pigs (Gabriel, 1979)

Title: Guinea Pig Contact Dermal Irritation/Sensitization

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar-diethyl-ar-methyl-, CAS No. 68479-98-1; Synonym: 

Diethyltoluenediamine (DETDA); Purity not specified

Remarks: Clear, light colored liquid; the typical composition of the commercial product is 75-

81% 3,5-diethyltoluene-2,4-diamine, 18-20% 3,5-diethyltoluene-2,6-diamine, 0.5-3% dialkylated 

m-phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Dermal Sensitization – Guinea Pigs

GLP compliant?: Not indicated

Species/strain: Guinea Pigs, strain not indicated

Sex (male/female/both): Not indicated

Number of animals/sex/dose: 6, sex not indicated

Dose/concentration: Six guinea pigs were treated with DETDA via intradermal injections 

administered every other day for a total of 10 injections. Ten sensitizing injections followed on 

the back and upper flanks. The first sensitizing injection consisted of 0.05 mL while the 

remaining 9 injections consisted of 0.1 mL each. Two weeks after the 10th injection, a challenge 

injection of 0.05 mL was administered. Sites were scored at 24 and 48 hrs after the last injection. 

An additional 4 guinea pigs were treated as described above with the positive control, 0.1% 

dinitrochlorobenzene in ethylene dichloride. 

Test condition: Not indicated

RESULTS

Endpoint value(s): Not sensitizing. 
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CONCLUSIONS

The test article was determined to be a primary skin irritant, but not a sensitizing agent in guinea 

pigs.

STUDY RELIABILITY

[3] Not reliable. Important study details missing from the study summary included purity and 

composition of the test substance, test method/guideline followed and findings/observations. 

REFERENCES

Gabriel, K. 1979. Guinea Pig Contact Dermal Irritation/Sensitization. Performing Laboratory: 

Biosearch, Inc. Submitted by the Ethyl Corporation, Baton Rouge, LA. As cited in Albemarle 

Corporation. 2011. Letter to EPA DCO dated May 4, 2011. OPPT-2007-0531, MR #335194.
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D-6. Toxicokinetic Studies

Study 1: Toxicokinetic study in Fischer 344 rats (Hainje and El-hawari, 1982)

Title: Absorption, distribution, elimination, and bioretention of 14C-labeled 

diethyltoluenediamine following repeated oral dosing in rats

TEST SUBSTANCE 

Identity: > 99% Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Referred to as 

Diethyl Toluene Diamine (DETDA)

Remarks: Technical grade (80:20 mixture of the 2,4- and 2,-6 diamino isomers); Lot 

XP213997A

METHOD

Method/guideline followed: Not indicated

Test type: Oral toxicokinetic study

GLP compliant?: Yes

Year study performed: 1981-1982

Test species: Fischer 344 rats (At the initiation of the studies, rats were 64-73 days old; the 

males weighed 158 to 228 g and the females weighed 102 to 154 g.)

Number of animals/sex/dose: 6

Route of administration: Oral

Vehicle: 0.05 N HCl

Exposure duration: 10 days

Doses/concentrations tested: 50 mg/kg-bw-bw

Statistical methods used: The means ± standard errors were calculated for each test group with 

a programmable (Monroe) calculator. The significance of the data was determined by the twotailed Student's t test. Significant differences were indicated when p < 0.05.

Test method/conditions remarks: The absorption, distribution elimination, and bioretention of 

technical grade diethyltoluenediamine (a mixture of the 2,4- and 2,6-diamino isomers) were 

examined in Fischer 344 rats treated with 10 daily doses (50 mg/kg-bw) of the 14C-labeled 

compound (14C-DETDA). Control rats received nine daily doses of the vehicle (0.05 N HCl) 
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followed by the tenth dose of 14C-DETDA. Levels of radioactivity in blood, urine, and feces 

were measured, and tissue levels were determined at 24 and 96 hr following the last dose. 

14C-DETDA was administered to 6 rats by gavage at a dose level of 50 mg/kg (= 1/10 of the 

LD50) as specified by the sponsor. A limited preliminary study was also performed in two rats 

treated with a 10 mg/kg dose of 14C- DETDA. The dosing solutions were prepared by dissolving 

the appropriate amounts of the nonlabeled and 14C-labeled DETDA in a volume of 5 ml/kg of 

0.05 N hydrochloric acid. Fresh dosing solutions were prepared for the preliminary and 

definitive studies in order to avoid any possible decomposition. Aliquots of each solution were 

counted to determine the amounts of radioactivity. The dosing solutions used in this study 

contained between 173 and 213 μCi of the labeled compound per 5 ml of the vehicle. When not 

in use, the dose was stared frozen under argon, The stability of the dosing solution was checked 

periodically by radio-gas chromatography.

Preliminary studies: The preliminary studies were performed with eight rats (four males and four 

females). Three males and three females were treated with single oral doses of 50 mg/kg 14CDETDA. Blood levels were examined during a 24-hr period in one male and one female rat. 

Another male and female rat were used to assess respiratory elimination of radioactivity during a 

48-hr period. The remaining male and female rats were used to assess urinary and fecal excretion 

of radioactivity during a 96-hr period. In addition, one male and one female rat were treated 

with a lower (10 mg/kg) dose of 14C-DETDA and were used to assess urinary and fecal excretion 

of radioactivity during a 96-hr period. At sacrifice, tissues of all rats were examined for residual 

radioactivity (see definitive studies).

For blood level determinations in the preliminary studies, samples were collected by tail bleeding 

at 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hr following dosing. During the experiment the rats were 

housed in individual stainless steel metabolism cages. Urine was collected at 4, 8, 12 and 24 hr, 

and feces were sampled at 12 and 24 hr, and then the rats were sacrificed.

Respiratory elimination of 14-CO2 or other volatile materials was assessed during 0-12, 12-24, 

and 24-48 hr intervals following dosing. The rats were housed individually in Delmar-Roth type 

glass metabolism cages. The CO2 trapping solution was 5 M ethanolamine in 2-methoxyethanol. 

For collection of other volatile products, methanol: water (50:50) was used. Urine and feces 

were sampled during the same intervals, and the animals were sacrificed at 48 hr. 

Urinary and fecal excretion were assessed during a 96-hr period in one male and one female rat 

treated with a 50 mg/kg dose, and one male and female treated with a 10 mg/kg dose of 14CDETDA. The rats were placed in stainless steel metabolism cages and urine was sampled 4, 8, 

12, 24, 48, 72 and 96 hr; feces were collected at 12, 24, 48, 72 and 96 hr following dosing, and 

then the rats were sacrificed.

Definitive studies: A total of 24 rats (12 males and 12 females) were divided into two groups of 

12 rats (6 males and 6 females) each. The rats in each group received either 10 daily doses of 

14C-DETDA or 9 daily doses of the vehicle followed by a tenth dose of 14C-DETDA. Daily 

dosing was performed between 8 and 9 a.m. After the last dose, the rats were placed in individual 

stainless steel metabolism cages. Three males and three females from each group were used for 
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blood level determinations. Blood samples were collected by tail bleeding at 0.5, 1, 2, 4, 8, 12, 

and 24 hr. To assess total recovery, urine and feces were also collected. The rats were sacrificed 

at 24 hr for tissue sampling (see below).

The remaining three rats/sex/group were used for assessment of urinary and fecal excretion of 

radioactivity. Urine was collected at 4, 8, 12, 24, 48, 72, and 96 hr. Feces were sampled at 12, 

24, 48, 72, and 96 hr following the last dose. After each collection the cages were rinsed and the 

cage washings sampled for radioactivity. The animals were sacrificed at 96 hr for analysis of 

radioactivity in tissues (see below).

The rats were sacrificed by withdrawal of blood from the abdominal aorta under light ether 

anesthesia and the following tissues and organs were removed, weighed, and analyzed for 

radioactivity: liver, kidneys, lungs, heart, spleen, pancreas, brain, thyroids, adrenals, gonads, 

bone marrow, urinary bladder, skeletal muscle, retroperitoneal fat, skin and GI tract plus 

contents.

Portions of blood were centrifuged; plasma and red blood cells (RBCs) were separated for 

radioactivity determinations. Prior to sampling, the RBCs were resuspended in 0.9% NaCl and 

recentrifuged. The urinary bladder contents were removed and the bladders were washed 

thoroughly with 10% ethanol before analysis.

Sample Preparation and Analysis: Aliquots (50 to 250 μ) of whale blood, plasma, and RBCs 

were analyzed for total radioactivity. Volumes of urine and cage rinse were measured and 

samples (250 μl) were analyzed. The trapping solutions for expired radioactivity were also 

measured, and samples (2 ml) were analyzed. Feces, GI tract, and tissues larger than 0.25 g were 

weighed and homogenized in five volumes of ethanol:water (10:90). Samples (60 to 120 mg) of 

other organs and tissues were weighed and assayed for 14C-DETDA content. Blood components, 

tissues, and fecal samples were combusted in a Packard Tricarb Sample Oxidizer (Model C306). 

Permafluor V® in combination with Carbo-Sorb® (Packard Instrument Company) was used as 

the scintillation cocktail. Urine and cage rinses were counted directly in Phase Counting Solution 

(PCS, Amersham).

Measurement of Radioactivity: Vials were cooled for a minimum of 24 hr before counting in a 

liquid scintillation counter (Packard Tricarb Model 3255). Correction for background was carried 

out automatically on the counter. Background determinations were obtained from the average of 

natural counts of several tissue homogenates from nontreated animals. The counting efficiency 

was determined using the automatic external standard (AES) method. An AES versus efficiency 

curve was prepared by processing a quench curve set through the counter under the conditions 

used throughout the experiment. Assays not within ± 10% of the mean of the duplicates were 

reassayed in duplicate except when the sample was no longer available o r when radioactivity 

counts were low and nonsignificant, i.e., less than two times the background, 30 cpm (counts per 

minute).

Data Processing and Analysis: 14C-DETDA contents in blood and tissues were presented in 

terms of microgram equivalents per milliliter (blood) or gram (tissues), and percentage of the 

dose administered to each animal. Microgram equivalents per milliliter of blood, or gram of 
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tissue were also presented in graphic form. Cumulative excretion in urine and feces was 

calculated and presented in tables as well as in graphic form. The means ± standard errors were 

calculated for each test group with a programmable (Monroe) calculator. The significance of the 

data was determined by the two-tailed Student's t test. Significant differences were indicated 

when p < 0.05.

The percent of administered dose recovered in blood, muscle, and skin was calculated based on 

7, 40, and 26%, respectively, of body weight. Percent recovery in plasma and RBCs was 

calculated based on 54 and 46%, respectively, of the total blood volume of each rat.

RESULTS

Preliminary Study: A single oral dose of 14C-DETDA was slowly absorbed and distributed to 

various organs and tissues. The compound and/or its metabolites were eliminated in urine and to 

a lesser extent in feces; excretion in expired air was negligible. Residual radioactivity in tissues 

was highest in thyroids, followed by liver, kidneys, and adrenals. Male rats demonstrated higher 

levels of 14C- in thyroids and liver than females.

Definitive Study: Following the last dose, rats treated with 10 doses of 14C-DETDA displayed 

significantly (up to 5-fold) higher levels of radioactivity in blood than rats treated with one dose. 

The females demonstrated higher 14C-DETDA levels in blood than their male counterparts. No 

significant differences in urinary excretion of radioactivity was apparent between rats of either 

treatment group. However, fecal excretion of was greater in rats treated with 10 doses as 

compared to those receiving 1 dose of 14C-DETDA. Female rats of both treatment groups 

eliminated higher amounts of 14C-DETDA in urine than their male counterparts.

At 24 and 96 hr after the last doses, the concentration of ra6ioactivity in tissues was highest in 

thyroids, followed by liver, kidneys, and adrenals. 14C-DETDA levels in skin were higher than in 

fat or muscle. Rats treated with 10 doses displayed significantly higher levels of 14C-DETDA in 

most tissues examined than those treated with one dose; the levels were up to 5-fold higher at 24 

hr and up to 6-fold higher at 96 hr. At 24 hr, females of both treatment groups showed 

considerably lower amounts of 14C-DETDA in liver but higher amounts in blood, RBCs, brain, 

spleen, and pancreas. At 96 hr, liver and thyroids of the females contained lower 14C-DETDA 

levels but differences in contents of other tissues were less apparent. Generally, disappearance of 

radioactivity from blood and tissues occurred at a faster rate in rats treated with 1 dose as 

compared to those receiving 10 doses of 14C-DETDA.

In rats treated with 10 doses of 14C-DETDA, recoveries of radioactivity at 24 hr in blood, 

muscle, skin, and tissues were 2- to 3-fold higher than in rats receiving one dose. Recoveries in 

the GI tracts did not differ significantly in the two treatment groups. At 36 hr, however, 

recoveries in the GI tracts as well as in blood, muscle, skin, and tissues were considerably (3-to 

6-fold) higher in the rats treated with 10 doses.

Results remarks: Since control rats (i.e., nontreated with DETDA) were not included in the 

studies, no attempt was made t o statistically analyze the changes in bodyweight and organ 

weight parameters. 
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CONCLUSIONS

The results of these studies indicate that 14C-labeled diethyl-toluenediamine (14C-DETDA) 

administered orally to rats was slowly absorbed and distributed to various organs and tissues. 

The compound and/or its metabolites were eliminated in urine and to a lesser extent in feces; 

excretion in the expired air was negligible. Radioactivity recovered in tissues at 24 and 96 hr 

after dosing was highest in the thyroids, followed by liver, kidneys, and adrenals. Levels of 

radioactivity in blood and tissues of female rats were generally higher than in males; 14C levels in 

liver and thyroids were higher in the males.

Treatment of Fischer 344 rats with 10 daily oral doses of the 14C-labeled compound resulted in 

significantly higher blood levels of 14C compared to those of rats receiving one dose (vehiclepretreated controls). Although the rates of urinary excretion were similar in both treatment 

groups and the rates of fecal excretion were higher in the rats pretreated with 14C-DETDA, the 

amounts of radioactivity retained in blood and tissue of the latter group were considerably higher 

than those of the vehicle-pretreated controls. If the retained radioactivity, however, is expressed 

as percentages of the total (10) administered doses, the amounts recovered in blood and tissue 

were lower than in the vehicle-pretreated controls.

The data obtained from these studies did not indicate an impairment of the excretory functions in 

rats pretreated with 14C-DETDA. Therefore, the retention of radioactivity in blood and tissues of 

rats receiving the 10 daily doses of the labeled compound suggests that the 14C level (probably 

from metabolites) binds to certain blood and tissue components. Definitive explanations of these 

findings require further assessment of the biotransformation and disposition of DETDA at 

several dose levels and its possible interaction with blood and tissue macromolecules.

STUDY RELIABILITY

[2] Reliable with restrictions. The test method/guideline followed was not specified.

REFERENCE

Hainje, M. and El-hawari, A. 1982. Absorption, Distribution, Elimination, and Bioretention of 

14C-Labeled Diethyltoluenediamine Following Repeated Oral Dosing to Rats. MRI Project No. 

7348-B. Performing Laboratory: Midwest Research Institute, Kansas City, MO. Sponsor: Ethyl 

Corporation, Baton Rouge, LA.
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Study 2: Toxicokinetic study in Sprague Dawley rats (Babin et al., 1996)

Title: The uptake and distribution of diethyltoluenediamine in the male Sprague Dawley rat

TEST SUBSTANCE 

Identity: Tritiated Benzenediamine, ar,ar-diethyl-ar-methyl, CAS No. 68479-98-1; Referred to 

as Diethyl Toluene Diamine (DETDA); Ethacure

Remarks: Ethacure is a mixture of compounds composed mostly of 2 major isomers, 2,4-

DETDA (75-81%) and 2,6-DETDA (18-20%). The test solution was tritiated 2,6-DETDA or 

2,4-DETDA diluted with non-tritiated Ethacure (84μmol/ml) in water to provide a labeled 

mixture with a specific activity of 0.295-0.48 μCi/μmol. 

METHOD

Method/guideline followed: Not indicated

Test type: Oral toxicokinetic study

GLP compliant?: Not indicated

Year study performed: 1996 (final report date)

Test species: Sprague Dawley rats (10-12 weeks, 159-296 g)

Number of animals/sex/dose: Preliminary study: 1 control male, 3 treated males; Tissue 

distribution study: 10 groups of 4 males; Elimination study: 10 males

Route of administration: Oral (gavage)

Vehicle: Tritiated 2,4-and 2,6-DETDA were diluted with non-tritiated Ethacure (84μmol/ml) in 

water

Exposure duration: Single dose

Doses/concentrations tested: 179 μmol/kg-bw

Statistical methods used: None specified

Test method/conditions remarks: Using synthesized acetylated DETDA standards, a 

preliminary rat study was performed to determine the extent of acetylation occurring with 

DETDA during metabolism. 

In the preliminary study, one group of 4 young adult (10-12 weeks) male Sprague Dawley 

rats (1 control and 3 treatment, 172-186 g) were administered a single dose of vehicle or the 
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commercial DETDA product in water at 179 μmol/kg bw. Urine was collected via 

metabolism cages (0-4, 4-8, 8-16 and 16-32 hrs). Urine samples were prepared for analysis 

using the solid phase extraction protocol. 

Distribution Study: Ten groups (5 per isomer) of 4 young adult male Sprague Dawley rats 

each were administered a single dose of tritiated 2,4- or 2,6-DETDA diluted with nontritiated Ethacure in water (to provide a labeled mixture with a specific activity of 0.46 

μCi/μmol) at179 μmol/kg bw) via gavage. Urine, feces, blood and tissues were collected at 

1, 4, 8, 24 and 48 hours post-dosing in individual metabolism cages. A day 6 tissue 

distribution time point was incorporated in the 6 day excretion study described below. Rats 

were anesthetized with isoflurane at each time point. Blood was collected via cardiac 

puncture at each time point. The following tissues were removed from each rat at the time of 

death: fat (epididymal fat pad), skin (ventral midline), skeletal muscle (right quadriceps), 

thymus, lung (right caudal lobe), heart (apex), spleen (caudal end), pancreas, bladder, kidney 

(right caudal area), liver (right caudal lobe), stomach (greater curvature), proximal 

duodenum, caecum (apex), colon (proximal ascending), thyroid, and brain (right frontal 

lobe). 

Excretion Study: Ten (5 per isomer) young adult male Sprague Dawley rats were 

administered a single dose of tritiated 2,4- or 2,6-DETDA diluted with non-tritiated 

Ethacure in water (to provide a labeled mixture with a specific activity of 0.48 μCi/μmol) at 

179 μmol/kg bw) via gavage. Urine and feces were collected from individual metabolism 

cages at 24 hour intervals over 6 days post-dosing. On day 6 rats were sacrificed and blood 

was collected via cardiac puncture. Contents from the stomach, small intestine, caecum, and 

large intestine were added to the day 6 time points. The following tissues were removed 

from each rat at the time of death for a day 6 distribution study: fat (epididymal fat pad), skin 

(ventral midline), skeletal muscle (right quadriceps), thymus, lung (right caudal lobe), heart 

(apex), spleen (caudal end), pancreas, bladder, kidney (right caudal area), liver (right caudal 

lobe), stomach (greater curvature), proximal duodenum, caecum (apex), colon (proximal 

ascending) and brain (right frontal lobe).

Covalent Binding Study: Three young adult male Sprague Dawley rats were administered a 

single dose of tritiated 2,4- or 2,6-DETDA diluted with labeled Ethacure (to attain a specific 

activity of 8.42 μCi/μmol) at 179 μmol/kg bw via gavage. Rats were sacrificed 24 hours 

after dosing at which time the livers were harvested, weighed and prepared for DNA 

extraction, nucleic acid and protein precipitation. 

RESULTS

In the preliminary study, only 2 of the 3 rats administered Ethacure were quantitatively evaluated 

because of the questionable dosage administered to one of the rats. In the first rat, 4.0% of the 

administered dose was recovered in the urine as both 2,4- and 2,6-DETDA (parent compound) 

by 32 hours. In the second rat, 4.4% of the administered dose was recovered as both 2,4- and 

2,6-DETDA. None of the expected acetylated DETDA metabolites were detected in the urine 

above 500 ng/ml (limit of quantitation) in any of the 3 rats treated. 
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The data showed very high levels of label occurred in the gastrointestinal system and bladder 

during the first 8 hours reflecting compound that was either unabsorbed, distributed or excreted. 

The metabolic and excretory organs, liver and kidney, also showed moderate levels. Label in the 

thyroid and pancreas was much higher than expected, for non-excretory organs, with thyroid 

levels exceeding; even that of liver tissue. The concentration of radioactivity in all tissues except 

the stomach, duodenum, caecum, colon, bladder and thyroid was greatest at 4 hours. With the 

exception of the stomach, duodenum, caecum, colon, bladder and thyroid, tissues exhibited a 

classic biphasic model d distribution and excretion with the onset of the slow phase occurring 

after the 4 hour time point. Thyroid levels, at 24 and 48 hours, were the highest of all tissues 

examined at that time point. As can be gleaned from the 24 and 48 hour time frames, DETDA 

was rapidly cleared from all tissues. Day 6 residual levels of label were fairly uniform in all 

tissues examined. Distribution levels for day 6 thyroid were unavailable, but the 48 hour time 

point values suggested that the thyroid levels at day 6 were probably elevated relative to other 

tissues.

The data revealed the highest levels of label occurred in the gastrointestinal system during the 

first 8 hours reflecting both absorbed and possibly unabsorbed 2,6-DETPA. Relatively high 

levels of radioactivity was present in the bladder for the first 8 hours presumably reflecting both 

distributed and excreted compound. The metabolic and excretory organs, liver and kidney, 

contained moderate levels of label in relatively equal amounts. Label in the thyroid and pancreas 

was much higher than expected, for nonexcretory organs, with thyroid levels exceeding even that 

of Fiver tissue. The concentration of radioactivity in all tissues except the stomach, caecum, 

colon, thyroid, pancreas and spleen was greatest at 4 hours. With the exception of the stomach, 

thyroid, pancreas and spleen, tissues exhibited a classic biphasic model of distribution and 

excretion with the onset of the slow phase occurring after the 4 hour time point. As can be seen 

from the 24 and 48 hour time frames, DETDA was rapidly cleared from all tissues. Day 6 

residual levels of label was uniform in all tissues except the thyroid gland which was almost 4 

fold higher than any other tissue.

These distribution profiles of labeled DETDA indicates the compound has both water and fat 

soluble properties. There appears to be little difference in the pattern of distribution between the 

2,4-DETDA and the 2,6-DETDA isomers with the possible exception of the thyroid gland.

Differences in the excretion profile between the 2,4- and 2,6-DETDA isomers seem to signify 

variations in the biotransformation of each compound. The greater urinary excretion of 2,6-

DETDA (58%) compared to 2,4-DETDA (44%) suggests a more excretable, water soluble 

metabolite may have been produced. This could be accomplished by greater phase II conjugation 

reactions (glucuronidation, sulfation, glutathione) on the 2,6-isomer compared to the 2,4-isomer. 

Restricted enzymatic access due to the flanking of the 4-position amino group by benzylic ethyl 

groups on the 2,4-isomer could effect these results.

Analysis of the data from the 24-hour time point urine samples revealed that 1 8.2% of labeled 

material was contained in the organic fraction. Acid hydrolysis demonstrated that 37.6% of the 

labeled material was in the form of acid-labile conjugates. The remaining 44.2% of the labeled 

material was composed of the non acid-labile water soluble fraction.
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Relative to Ethacure (80.8% 2,4-DETDA, 19.2% 2,6-DETDA), 16 pmol of 2,4-DETDA/μmol of 

DNA and 5 pmol of 2,6-DETDA/μmol of DNA was found covalently bound. For the 2,4-

DETDA isomer, 55% of the label was bound to the protein fraction equating to 108 nmol 2,4-

DETDA per gram of liver protein. With the 2,6-DETDA isomer, 38% of the label was bound to 

the protein fraction equating to 31 nmol 2,6-DETDA per gram of liver protein. These results 

suggest that compared to TDA, both 2,4- and 2,6-DETDA are less likely to be involved in 

covalent binding to DNA.

Results remarks: None specified

CONCLUSIONS

DETDA is absorbed after oral administration, and eliminated in the urine and feces. The 

presence of conjugated metabolites in urine was indicated; however, alkylated metabolites were 

not detected in urine. Roughly equivalent concentrations, on a μmol/g basis, were detected in 

the thyroid, pancreas and liver.

STUDY RELIABILITY

[2] Reliable with restrictions. Details missing from the study included test substance purity, 

method/guideline followed and GLP compliance.

REFERENCE

Babin, M.C., Swenson, D.H., Short, C.R., and Barker, S.A. 1996. The Uptake and Distribution 

of Diethyltoluenediamine in the Male Sprague Dawley Rat. LSU School of Veterinary 

Medicine. Baton Rouge, LA. Sponsor: Albemarle Corporation, Baton Rouge, LA.
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E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information 

Available

Acceptable Data Gap

Aquatic Effects

Acute Toxicity to Fish No – Yes

Acute Toxicity to Daphnia No – Yes

Toxicity to Aquatic Plants (Algae) Yes Yes No

Health Effects

Acute Toxicity Yes Yes No

Repeated-Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity No – Yes

Developmental Toxicity No – Yes