SRC TR

- 1 4

- 038

HPV2 TEST RULE DATA ADEQUACY REVIEW : SEPTEMBER 2014 UPDATE ( FINAL )

Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1

Prepared by:

Ashlee Aldri dge

, Amy Nguyen, Justine Von Runnen, Chris topher Janson, Julie Ramage, Julie Klotzbach, Jennifer Rayner

, Kelly Salinas, David Wohlers, Teresa Manyin

, and William L. Richards

Defense and Environmental Solutions Division

SRC, Inc. 7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027 Task FG007. 4 .009.001

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue Washington, D.C. 20460

Bryan Lobar , Project Officer David Brooks

, Work Assignment Manager Amy Benson , Alternate Work Assignment Manager

September 12

, 201 4Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1
B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 2

C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 2

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 4

D

- 1. Acute Toxicity to Fish

................................ ................................ ................................ ....... 4

D

- 2. Acute Toxicity to Aquatic Invertebrates

................................ ................................ ............ 8

D

- 3. Toxicity to Aquatic Plants

................................ ................................ ............................... 12

D

- 4. Acute Toxicity

................................ ................................ ................................ .................. 16

D

- 5. Repeated

- Dose Toxicity

................................ ................................ ................................ ... 22

D

- 6. Genetic Toxicity

– Gene Mutation

................................ ................................ ................... 43

D

- 7. Genetic Toxicity

– Chromosomal Aberrations

................................ .............................. 123

D

- 8. Reproductive Toxicity

................................ ................................ ................................ .... 133

D

- 9. Developmental Toxicity

................................ ................................ ................................ . 134
E. SUPPLEMENTAL DATA (NON

- SIDS ENDPOINTS)

................................ ...................... 142

E

- 1. Genetic Toxicity

– Other

................................ ................................ ................................ 142

E

- 2. Carcinogenicity

................................ ................................ ................................ ............... 170

E

- 3. Skin Irritation

................................ ................................ ................................ .................. 184

E

- 4. Eye Irritation

................................ ................................ ................................ ................... 186

E

- 5. Skin Sensitization

................................ ................................ ................................ ........... 188

E

- 6. Toxicokinetic Studies

................................ ................................ ................................ ..... 190
F. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ......... 200Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document provides a review of data describing the toxicity of benzenediamine, ar, ar

diethyl

- ar

- methyl

- (CASRN 68479

- 98

- 1) . In addition to data that were previously assessed for adequacy by EPA 1 , this document evaluates a newly submitted prenatal developmental toxicity study for CASRN 68479

- 98

- 1 , which was submitted on August 2, 2014 by Albemarle Corporation and Lonza Inc. to 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 ; available online at http://www.regulations.gov/# !documentDetail; D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0579 as of September 12 , 2014 ).

The HPV2 test rule requires the following toxicological testing for CASRN 68479

- 98

- 1 : C1 (consisting of Test Group 1 [acute toxicity to fish, acute toxicity to Daphnia , and toxici ty to algae tests] if log K ow < 4.2 or Test Group 2 [chronic toxicity to Daphnia and toxicity to algae tests] if log K ow ≥ 4.2), E1 (bacterial reverse mutation test), E2 (chromosomal aberration s or micronucleus test), and F1 (combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test). Based on a measured log K ow of 1.38 2 , Test Group 1 testing is required to determine the toxicity of CASRN 68479

- 98

- 1 to aqu atic organisms.

Previous submissions in response to the toxicity testing requirements included the following:

(1) On May 4, 2011, Albemarle Corporation and Lonza Inc. supplied 22 full study reports for the following toxicological endpoints: toxicity to alg ae (component of C1), bacterial reverse mutation (E1), chromosomal aberration s (E2)

, and repeated

- dose toxicity (component of F1) ( partial submission available online at http://www. regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014). In addition to these studies, the sponsor also submitted 16 fu ll study reports and 6 brief study summaries to provide supplemental data to EPA for

non

- required endpoints.

(2) On March 1, 2012, Albemarle Corporation and Lonza Inc. supplied additional data in response to the mammalian cell mutation (E1) and the chromosomal aberration (E2) testing requirements ( submission available online at http://www.regulations.gov/ #!documentD etail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0660 as of September 12 , 2014).

(3) On June 29, 2012, Albemarle Corporation and Lonza Inc. supplied data for the acute toxicity to fish and the acute toxicity to Daphnia (component s of C1) testing

1 Data for CASRN 68479

- 98

- 1 were previously reviewed in the following report: “HPV2 Test Rule Data Adequacy Review; November 2012 Update: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1” (SRC Technical Report #12

- 081, dated November 5, 2012; av ailable online at http://www.regulations.gov/#!documentDetail; D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0706 as of September 12 , 2014).

2 The log K ow was experimentally determine d by Wildlife International Ltd. (Schutt, W.R., and Nixon, W.B. 2012. Determination of the n

- octanol/water partition coefficient of diethyltoluene diamine (DETDA) by the shake flask method. Wildlife International Ltd. Project No. 471C

- 143.) Study sub mitted by Albemarle Corporation to EPA on June 29, 2012 and available online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0677 as of Sep tember 12 , 2014.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

2 requirements (submission available online at http://www.regulations.gov/#!document Detail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0677 as of September 12 , 2014).

EPA evaluated the above su bmissions and determined that the available data were adequate to satisfy the C1, E1, and E2 testing requirements and the repeated

- dose toxicity component of the F1 testing requirement. Although no reproductive toxicity studies were submitted, since no hi stopathological effects in the reproductive organs were observed in the repeated

- dose toxicity studies, EPA agreed to waive the reproductive toxicity component of the F1 testing requirement, provided that the sponsors supply an adequate prenatal developmen tal toxicity test.

On August 2, 2014, Albemarle Corporation and Lonza Inc. supplied the following study report to complete their obligation for F1 testing:

 Theuns

- Van Vliet, J.G., and Otto, M. 2014. An oral prenatal developmental toxicity study with d iethylmethylbenzene diamine (DETDA) in rats. Performing Laboratory: TNO Triskelion, The Netherlands. TNO Triskelion Study No. 20193. Sponsors: Albemarle (Belgium), Bayer MaterialScience AG (Germany), and Lonza AG (Switzerland). Study report not availab le online as of September 12 , 2014.

Data from all studies submitted by Albemarle Corporation and Lonza Inc. are summarized and evaluated for adequacy in Section D. The newly submitted prenatal developmental toxicity study is evaluated on pp. 13 4

- 141 of this review. Additional data for non

- SIDS toxicity endpoints (“supplemental” data) are reviewed in Section E.

B. EVALUATING DATA ADEQUACY

Each study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details were compared to current standard test guidelines to establish whether the study met current testing requirements. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data availability/adequacy for each SIDS endpoint is included on the last page of this document (Section F).

C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

Evaluation of Toxicological Data Submitted on August 2, 2014: The prenatal developmental toxicity study

, submitted by Albemarle Corporation and Lonza Inc. on August 2, 2014, is adequate to satisfy the developmental toxicity component of the F1 testing requirement for CA SRN 68479

- 98

- 1 promulgated in the final HPV2 test rule.

Evaluation of Previously Submitted Toxicological Data: Toxicological data supplied by Albemarle Corporation and Lonza Inc. from May 4, 2011 to June 29, 2012 are adequate to satisfy the acute toxici ty to fish (component of C1), acute toxicity to Daphnia (component of C1), toxicity to algae (component of C1), bacterial reverse mutationFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

3 (E1)

, chromosomal aberration (E2) , and repeated

- dose toxicity (component of F1) testing requirements promulgated in the final HPV2 test rule.

Although an adequate reproductive toxicity study is not available, t he reproductive toxicity endpoint (component of F1) has been acceptably addressed on the basis that an adequate developmental toxicity study is available and n o treatement

- related histopathological effects in reproductive organs were observed at sublethal dietary concentrations of CASRN 68479

- 98

- 1 in adequate repeated

- dose toxicity studies and a two

- year cancer study

. Summary of Data Adequacy for Toxicolog ical Endpoints: Adequate data are available to satisfy all HPV2 toxicity testing requirements for CASRN 68479

- 98

- 1. There are no outstanding HPV test rule requirements for toxicity testing of CASRN 68479

- 98

- 1.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

4 D . DATA FOR SIDS ENDPOINTS

D

- 1

. Acute Toxicity to Fish

Summary of endpoint: Adequate

Study 1: Acute toxicity study in fathead minnow (Brougher et al. 2012a)

Title: Diethyltoluene Diamine (DETDA): A 96

- Hour Static Acute Toxicity Test with the Fathead Minnow ( Pimephales promelas )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1 ; Synonym: d iethyltoluene diamine (DETDA)

Remarks: Liquid; Lot No. 4500051816

- 011110; Composition: 97.3% active i ngredient (DETDA) , 1.27% dialkyl

- m

- phenylene diamines, 1.06% trialkyl

- m

- phenylene diamines, and 0.39% unknowns.

METHOD

Method/guideline followed: OECD TG 203 (1992) Fish, Acute Toxicity Test ” ; OPPTS 850.1075 (1996) “ Fish Acute Toxicity Test, Freshwater and Marine ” ; and ASTM E729

- 96 (2007) “ Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates, and Amphibians ”

. Test type: Acute Toxicity to Fish ( Static Conditions )

GLP compliant?: Yes, with the following exceptions: “Periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard US EPA analytical methods.” [Brougher et al. 2012a, p. 2]

Year study was performed: 2012

Test species: Fathead minnow ( Pimephales promelas ) ; obtained as juveniles from Chesapeake Cultures of Hayes, VA.

Analytical monitoring: Yes. “Samples were collected from each test chamber of each treatment and control group at the beginning of the test and at 48 and 96 hours (± 1 hour) to measure concentrations of the test substance. The samples were collected at mid

- depth, placed in g lass vials, and processed immediately for analysis

... The method used for the analysis of DETDA in the freshwater samples was based upon methodology developed by WildlifeFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

5 International, Ltd. The analytical method consisted of diluting the samples with fres hwater, as necessary, and analyzing by high performance liquid chromatography with variable wavelength detection at 220 nm. ” [ Brougher et al. 2012a, p. 11]

Exposure duration: 96 hours

Concentrations tested: Nominal concentrations: 7.5, 15, 30, 60, and 120 mg a.i./L; M ean m easured concentrations: 7.2, 15, 29, 70, and 106 mg a.i./L. Measured concentrations ranged from 74 to 139% of nominal concentrations.

Test method/conditions remarks: “Fathead minnows were exposed to a geometric series of five test concentrations and a negative control (dilution water) for 96 hours under static conditions. Two replicate test chambers were maintained in each treatment and control group, with 10 fish in each test chamber, for a total of 20 fish per concentration. Nom inal test concentrations were selected in consultation with the Sponsor based on the results of exploratory range finding toxicity data. Nominal test concentrations selected were 7.5, 15, 30, 60 and 120 mg Diethyltoluene Diamine (DETDA) active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning, the approximate mid

- point and the end of the test. Fathead minnows were impartially assigned to expos ure chambers at test initiation. Observations of mortality and other signs of toxicity were made approximately 2, 24, 48, 72 and 96 hours after test initiation. Cumulative percent mortality observed in the treatment groups was used to determine LC 50 values at 24, 48, 72 and 96 hours. The no

- mortality concentration and the no

observed

- effect concentration (NOEC) [for signs of toxicity] were determined by visual interpretation of the mortality and biological observation data.” [Brougher et al. 2012a, p. 8]

GROWTH MEDIUM AND DILUTION WATER: “The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wildlife International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 μm, and pumped into a 37,800

- L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filtered to 0.45 μm to remove fine particles.” [Brougher et al. 2012a, p. 10]

TEST SPECIES:

“ All fish used in the test were juveniles from the same source and year class, and the length of the longest fish measured was no more than twice the length of the shortest. The average total length of 10 negative control fish measured at the end of the te st was 3.0 cm, with a range of 2.8 to 3.2 cm. The average wet weight (blotted dry) of 10 negative control fish measured at the end of the test was 0.27 grams, with a range of 0.23 to 0.32 grams. Loading was defined as the total wet weight of fish per lit er of test water and was 0.18 g fish/L.” [Brougher et al. 2012a, p. 9]

“The fish were held for at least 14 days prior to the test in water from the same source and at approximately the same temperature as used during the test. Daily during the holding period, except during periods of fasting prior to testing, the fish were fed a commercially

- prepared diet supplied by Sera, North America, supplemented with brine shrimp nauplii ( Artemia sp.) supplied by Brine Shrimp Direct, Ogden, Utah. The fish were not fed for at least two days prior to the testFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

6 or during the test. During the 2

- week period prior to the test the fish showed no signs of disease
or stress. At test initiation, fathead minnows were collected from the culture tank and impartially distribute d one and two at a time to the test chambers until each contained 10 fish.” [Brougher et al. 2012a, p. 10]

PREPARATION OF TEST ITEM CONCENTRATIONS: “Individual test solutions were prepared directly in each test chamber. All test solutions were adjusted t o 100% active ingredient during preparation, based on the test substance purity (97.3%). Solutions were prepared at nominal concentrations of 7.5, 15, 30, 60 and 120 mg a.i./L by mixing calculated amounts of test substance into dilution water with a final volume of 15 L. The test substances were weighed into tared 100 mL glass beakers. Approximately 60 mL of the
pre

- measured 15 L dilution water was added, and the beakers were sonicated for 2

– 40 minutes. Using a portion of the pre

- measured dilution wat er, each test solution was then rinsed into its appropriate 25 L test chamber, and stirred with a top down mixer for 2

- 3 minutes. The negative control solution was dilution water only.” [Brougher et al. 2012a, p. 11]

EXPOSURE CONDITIONS: “Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness.” [Bro ugher et al. 2012a, p. 11 ] “ Light intensity at test initiation was 695 lux at the surface of the water of one representative test chamber.” [ Brougher et al. 2012a, p. 14 ]

“The target test temperature during the study was 22 ± 1oC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24

- hour intervals during the test using a liquid

- in

- glass thermometer. Temperature also was monitored continuously in one negative control test chamber.” [Brougher et al. 2012a, p. 13 ] “ Water temperatures were within the 22 ± 1oC range established for the test. ” [Brougher et al. 2012a, p. 14 ]

“Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at approximately 24

- hour intervals during the test.” [Brougher et al. 2012a, p. 13]

“ Dissolved oxygen concentrations remained ≥6.1 mg/L (≥70% of saturation) throughout the test. Measurements of pH ranged from 8.2 to 8.4 during the test.” [Brougher et al. 2012a, p. 14 ]

Hardness, alkalinity

, and specific conductance in the dilution water, which were measured at the beginning of the test, were 136 mg/L (as CaCO 3 ), 17 2 mg/L (as CaCO 3 ), and 315 μS/cm, respectively.

RESULTS

Endpoint value(s): 96

- hour LC 50 > 106 mg a.i./L; no

- mortality concentration = 15 mg a.i./L; NOEC (for signs of toxicity) = 15 mg a.i./L

Results remarks: Results of the study were based on mean measured concentrations. No mortality was observed in the control, 7.2, 15, or 7 0 mg a.i./L g roups after 96 hours of exposure. In the 29 and 1 0 6 mg a.i./L groups, 1/20 and 4/20 mortalities were observed after 96 hours,Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

7 respectively. Signs of toxicity, including lethargy, loss of equilibrium, and surfacing, were observed at ≥29 mg a.i./L. Regard ing determination of the toxicity values, “in this study, there was less than 50% mortality in any of the Diethyltoluene Diamine (DETDA) treatment groups during the test which precluded the statistical calculation of LC 50 values at 24, 48, 72 and 96 hours. Due to the method used to calculate the 96

- hour LC 50 value, the slope of the dose response curve could not be calculated. Since there was <50% mortality at 24, 48, 72 and 96 hours, the 96

- hour LC 50 value, as well as the no

- mortality concentration and NOEC, were determined by visual interpretation of the mortality and observation data.” [Brougher et al. 2012a, p. 13]

CONCLUSIONS

“Fathead minnows ( Pimephales promelas ) were exposed for 96 hours under static conditions to five mean measured concentrations of Diethyltoluene Diamine (DETDA) ranging from 7.2 to 106 mg a.i./L. The 96

- hour LC 50 value was >106 mg a.i./L, the highest concentration tested. Fathead minnows exposed to Diethyltoluene Diamine (DE TDA) at concentrations ≤15 mg a.i./L appeared normal with no mortality or overt signs of toxicity. The 96

- hour no

- mortality concentration and the NOEC value for signs of toxicity were 15 mg a.i./L .” [Brougher et al. 2012a, p. 15]

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCE S

Brougher

, D

. S

. , Kendall

, T

. Z

. , Gallagher

, S

. P

. , and Krueger

, H

. O. 2012a. Diethyltoluene Diamine (DETDA): A 96

- Hour Static Acute Toxicity Test with the Fathead Minnow ( Pimephales promelas ). Project No. 471A

- 123. Performing Laboratory: Wildlife International, Ltd. Sponsor: Albemarle Corporation, Baton Rouge, LA. Study report a vailable online at http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0677 as of September 12 , 2014.

Text in quotes i s taken directly from Brougher et al. ( 2012a )

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

8 D

- 2

. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: Adequate

Study 1: Acute toxicity study in Daphnia magna (Brougher et al. 2012b)

Title: Diethyltoluene Diamine (DETDA): A 48

- Hour Static Acute Toxicity Test with the Cladoceran ( Daphnia magna )

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1 ; Synonym: d iethyltoluene diamine (DETDA)

Remarks: Liquid; Lot No. 4500051816

- 011110; Composition: 97.27% active ingredient (DETDA)

, 1.27% dialkyl

- m

- phenylene diamines, 1.06% trialkyl

- m

- phenylene diamines, and 0.39% unknowns.

METHOD

Method/guideline followed: OECD TG 202 (2004) “ Daphnia sp., Acute Immobilization Test ” ; OPPTS 850.1010 (1996) “ Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids ” ; and ASTM E729

- 96 (2007) “ Standard Guide for Conducting Acute Toxicity Tests on Test Materials with Fishes, Macroinvertebrates and Amphi bians ”

. Test type: Acute T oxicity to Daphnia ( Static Conditions )

GLP compliant?: Yes, with the following exceptions: “Periodic analyses of water for potential contaminants were not performed according to Good Laboratory Practice Standards, but were performed using a certified laboratory and standard US EPA analytical methods. The stability of the test substance under conditions of storage at the test site were not determined in compliance with GLP standards.” [Brougher et al. 2012b, p. 3]

Year study was performed: 2012

Test species: Water flea ( Daphnia magna ), neonates < 24 hours, not first brood progeny, at test start; obtained from cultures maintained by the testing facility

. Analytical monitoring: Yes. “Samples were collected from the batches of test solution prepared for each treatment and control group at the beginning of the test and from each test chamber at 48 hours (± 1 hour) to measure concentrations of the test substance. The samples (~20 mL) were collected at mid

- depth, placed in glass vials, and processed immediately for analysis. The method used for the analysis of DETDA in the freshwater samples was based upon methodology developed by Wildlife International, Ltd. The analytical method consisted ofFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

9 diluting the samples with freshwater, as necessary, and analyzing by high performance liquid chromatography with variable wavelength detection at 220 nm. ” [Brougher et al. 2012b, p. 12]

Exposure duration: 48 hours

Concentrations tested: N ominal concentrations: 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0 mg a.i./L; M ean m easured concentrations: 0.24, 0.49, 0.98, 1.9, 3.9, and 7.8 mg a.i./L. Measured concentrations ranged from 95 to 98% of nominal concentrations.

Test method/conditions remarks: “Daphnids were exposed to a geometric series of six test concentrations and a negative control (dilution water) for 48 hours under static conditions. Two replicate test chambers were maintained in each treatment and control group, with 10 daphnids in each test chamber, for a total of 20 daphnids per concentration. Nominal test concentrations were selected in consultation with the Sponsor based on the results of exploratory range finding toxicity data. Nominal test concentrations selected were 0.25, 0.5, 1.0, 2.0, 4.0 and 8.0 mg Diethyltoluene Diamine (DETDA) active ingredient (a.i.)/L. Mean measured test concentrations were determined from samples of test water collected from each treatment and control group at the beginning and end of the test. Daphnid s were impartially assigned to exposure chambers at test initiation. Observations of immobility and other signs of toxicity were made approximately 2.5, 24 and 48 hours after test initiation. Cumulative percent immobility observed in the treatment groups was used to determine EC 50 values at 24 and 48 hours. The no

- immobility concentration and the no

- observed

- effect concentration (NOEC) [for signs of toxicity] were determined by visual interpretation of the immobility and biological observation data.” [B rougher et al. 2012b, p. 9]

TEST SPECIES: Name: Daphnia magna Maintenance and Acclimatization: Parental Daphnia were cultured in water from the same source and at approximately the same temperature as used during the test. First brood progeny were not used. The Daphnia were < 24 hours old at the start of the study.

GROWTH MEDIUM AND DILUTION WATER: “The water used for culturing and testing was freshwater obtained from a well approximately 40 meters deep located on the Wild life International, Ltd. site. The well water was passed through a sand filter to remove particles greater than approximately 25 μ m, and pumped into a 37,800

- L storage tank where the water was aerated with spray nozzles. Prior to use, the water was filte red to 0.45 μ m to remove fine particles and was passed through an ultraviolet (UV) sterilizer.” [Brougher et al. 2012b, p. 11]

PREPARATION OF TEST ITEM CONCENTRATIONS: “A primary stock solution was prepared by mixing a calculated amount of test substance in 2.0 L of dilution water at a nominal concentration of 8.0 mg a.i./L, the highest concentration tested. The stock solution was adjusted to 100% active ingredient during preparation, based on the test substance purity (97.3%). The stock solution was so nicated for approximately 2 minutes and mixed by inversion at least 20 times. Aliquots of the primary stock solution were proportionally diluted with dilution water to prepare five additional test solutions at nominalFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

10 concentrations of 0.25, 0.5, 1.0, 2.0 , and 4.0 mg a.i./L. The solutions were mixed by inversion and approximately 250 mL of solution was placed in each of two replicate test chambers per treatment group. The negative control solution was dilution water only.” [Brougher et al. 2012b, p. 12]

EXPOSURE CONDITIONS:

“ Ambient laboratory light was used to illuminate the test systems. Fluorescent light bulbs that emit wavelengths similar to natural sunlight were controlled by an automatic timer to provide a photoperiod of 16 hours of light and 8 hours of darkness.” [Brougher et al. 2012b, p. 13] “ Light intensity at test initiation was 617 lux at the surface of the water of one representative test chamber.” [Brougher et al. 2012b, p. 15 ]

The target test temp erature during the study was 20 ± 1oC. Temperature was measured in each test chamber at the beginning and end of the test and at approximately 24 hours during the test. Temperature also was monitored continuously during the test in a container of water p laced adjacent to the test chambers. “Water temperatures were within the 20 ± 1oC range established for the test.” [ Brougher et al. 2012b, p. 15 ]

“Dissolved oxygen and pH were measured in each test chamber at the beginning and end of the test and at appr oximately 24 hours during the test.” [Brougher et al. 2012b, p. 14] “ Dissolved oxygen concentrations remained ≥8.5 mg/L (≥93% of saturation) throughout the test. Measurements of pH ranged from 8.1 to 8.5. ” [ Brougher et al. 2012b, p. 15 ]

Hardness, alkalin ity, specific conductance

, and total organic carbon (TOC) in the dilution

w ater, which were measured at the beginning of the test, were 140 mg/L as CaCO 3 , 180 mg/L as CaCO 3 , 340 μS/cm, and <1 mg C/L, respectively.

RESULTS

Endpoint value(s): 48

- hour EC 50 = 5.8 mg a.i./L (based on mean measured concentrations); 95% Confidence Interval = 4.7

- 7.6 mg a.i./L; No

- immobility concentration = 1.9 mg a.i./L; NOEC (for signs of toxicity) = 0.98 mg a.i./L.

Results remarks: “ Daphnia in the negative control groups ap peared normal throughout the test. All daphnids in the 0.24, 0.49, and 0.98 mg a.i./L treatment groups also appeared normal throughout the test, with no immobility or overt signs of toxicity observed. Percent immobility at test termination in the 3.9 and 7.8 mg a.i./L treatment groups was 25 and 70%, respectively. Three daphnids in the 1.9 mg a.i./L treatment group appeared lethargic at test termination, while the remaining daphnids in this treatment group appeared normal throughout the test. Lethargic da phnids were observed in the 3.9 mg a.i./L treatment group at test termination, and lethargic daphnids were observed in the 7.8 mg a.i./L treatment group at both the 24

- hour interval and at test termination. The no

- immobility concentration was 1.9 mg a.i./ L and the NOEC [for signs of toxicity] was 0.98 mg a.i./L. EC 50 values at 24 and 48 hours were determined from the immobility data.” [Brougher et al. 2012b, pp. 15

- 16]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

11 CONCLUSIONS

“The cladoceran, Daphnia magna , was exposed for 48 hours under static con ditions to six mean measured concentrations of Diethyltoluene Diamine (DETDA) ranging from 0.24 to 7.8 mg a.i./L. The 48

- hour EC 50 value was 5.8 mg a.i./L, with a 95% confidence interval of 4.7 to 7.6 mg a.i./L. The slope of the concentration

- response curve was 4.616. The no

- immobility concentration was 1.9 mg a.i./L and the NOEC [ for signs of toxicity ] was 0.98 mg a.i./L.” [Brougher et al. 2012b, p. 16]

STUDY RELIABILITY

[1] Reliable without restrictions

.

REFERENCE S

Brougher

, D

. S

. , Kendall

, T

. Z

. , Gallagher

, S

. P

. , and Krueger

, H

. O. 2012b. Diethyltoluene Diamine (DETDA): A 48

- Hour Static Acute Toxicity Test with the Cladoceran ( Daphnia
magna ). Project No. 471A

- 122. Performing Laboratory: Wildlife International, Ltd. Sponsor: Albemarle Corporation, Baton Rouge, LA. Study report a vailable online at http://www. regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0677 as of September 12

, 2014.

Text in quotes i s taken directly from Brougher et al. ( 2012b )

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12 D

- 3 . Toxicity to Aquatic Plants

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

- , CASRN 68479

- 98

- 1; Synonym: Diet hyltoluenediamine (DETDA) ; Referred to as DETDA (Diethyltoluylendiamin

- 80 ) and Diethylmethylbenzoldiamine in the study report.

Remarks: C lear, light

- colored liquid; Batch No. HOTU 241770

- 7; Purity: 97.7%; Composition: 2,4

- DETDA ( 78.5 %), 2,6

- DETDA ( 19.2 %), 2,4,6

- TEMPDA ( 0.5 %) , METDA (1%), Total TAPDA (0.56%)

.

METHOD

Method/guideline followed: OECD TG 201 (2006) “Alga, Growth Inhibition Test” and M ethod C.3 of Commission Regulation (EC) No. 761/2009 (2009) “Freshwater Alga e and Cyanobacteria, Growth Inhibition Test”

Test type: Toxicity to Plants

– Algae (Static Conditions)

GLP compliant?: Yes

Year study was performed: 2010

Test species: Freshwater green algae ( Desmodesmus subspicatus , formerly Scenedesmus subspicatus ) ; obtained from The Collection of Algal Cultures of the Institute of Plant Physiology at the University of G ö ttingen (Germany).

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 values 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,Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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13 1.0, 3.2, 10, 32, and 100 mg/L of DETDA dissolved in dilution water. The cell densities were measured at 24

- hour intervals. Inhibition of algal population wa s measured as reduction in growth rate relative to control cultures. The 72

- hour EC 10 and EC 50 were calculated by probit analysis. All calculations were carried out using the statistics program ToxRatPro Version 2.10 (released 2010

- 02

- 20). For the calc ulations

, 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 t est 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 beginni ng 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. ” [Caspers 2010, p. 9]

Test Species : Desmodesmus subspicatus Strain No. 86.81 SAG

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 s - 1 (measured in the range 400 to 700 nm using a spherical quantum 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. ” [Caspers 2010, p. 12]

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. ” [Caspers 2010, p. 12]

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. ” [Casp ers 2010, p. 12]

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. ” [Caspers 2010, p. 12]

Pre

- treatme nt 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 a n ultra turrax and afterwards stirred for 24 h on a magnetic stirrer. The pH was measured to be 8.0. ” [Caspers 2010, p. 14]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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14 “ 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. ” [Caspers 2010, p. 14]

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 r ange 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. ” [Caspers 2010, p. 14]

“ 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. ” [Caspers 2010, p. 14]

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 /mL

. 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 CaCO 3

.

Evaluation of Cell Densities: “Cell densities were measured in a microcell counter by taking small aliquots from each test flask for measurements.” [Caspers 2010, p. 15]

RESULTS

Endpoint value(s): Based on nominal concentrations:

Growth Rate: 72

- hour EC 50 = 104 mg/L (95% confidence interval: 102

- 106 mg/L) 72

- hour EC 10 = 54 mg/L 72

- hour NOEC = 32 mg/L 72

- hour LOEC = 100 mg/L

Yield: 72

- hour EC 50 = 60 mg/L (95% confidence interval: 58

- 62 mg/L) 72

- hour EC 10 = 37 mg/L 72

- hour NOEC = 10 mg/L 72

- hour LOEC = 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. ” [Caspers 2010, p. 10]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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15 “ 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%. ” [Caspers 2010, p. 10]

“ The coefficient of variation of average specific grow th rates during the whole test period in replicate control cultures did not exceed 7%. ” [Caspers 2010, p. 10]

CONCLUSIONS

Based on nominal concentrations, t he 72

- hr EC 50 was 104 mg/L for growth rate and 60 mg/L for yield. The 72

- hour NOEC and LOEC for growth rate 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.

REFERENCE S

Caspers. 2010. Alga, growth inhibition with DETDA (Diethyltoluylendiamin

- 80)

. Study No. 2010/0109/01. Performing Laboratory: CURRENTA GmbH & Co. OHG. Leverkusen, Germany. Sponsor: Albemarle Europe SPRL , Louvain

- la

- Neuve, Belgium.

Text in quotes i s taken directly from Caspers ( 2010 )

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16 D

- 4 . Acute Toxicity

Summary of endpoint: Adequate

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

Title: Acute Oral Toxicity Study

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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% dial kylated m

- phenylenediamines and < 0.08% water.

METHOD

Method/guideline followed: Not indicated

Test type: Acute Oral Toxicity

-- 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 and 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. ” [Albemarle Corporation 2011, p. 11]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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17 RESULTS

Endpoint value(s): LD 50 = 485 mg/kg

- bw (95% confidence interval: 406

- 564 mg/kg

- bw)

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

CONCLUSIONS

“ The oral LD 50 was estimated to be 485 mg/kg with 95% confidence limits of 406 and 564 mg/kg. ” [Albemarle Corporation 2011, p. 11]

STUDY RELIABILITY

[2] Reliable with restrictions. Missing details included test method/guideline followed, purity/composition of the tested commercial product, mortality incidence, clinical observations/ findings, and whether or not body weight data were recorded.

REFERENCES

Heenehan, P., Braun, W., and Rinehart, W. 1977 a

. Acute Oral Toxicity Study. Compound: Diethylated Toluenediamine. Project No. 4448

- 77. Performing Laboratory: Bio/dynamics Inc. Sponsor: Ethyl Corporation, Baton Rouge, LA. [ As summarized in Albemarle Corporation, 2011. ]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011 )

. Submission a vailable online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of Septem ber 12 , 2014.

Text in quotes i s taken directly from Albemarle Corporation (2011).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

18 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 singl e concentration

, which did not induce any mortalities

, 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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 Toxicity

-- 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 specifiedFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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19 Test method/conditions remarks: “ Sprague Dawley rats (5/sex/dose) were exposed for 1 hour to a nominal exposure concentration of 2.45 mgDETDA/L. No additional details were available from this study summary. ” [ Albemarle Corporation 2011, p. 11]

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 disappe ared during the 4 hr period post

- dosing. Lung discoloration was observed at necropsy on d 14. ” [ Albemarle Corporation 2011, p. 11]

Endpoint value(s): L C 50 (1

- hr) > 2.45 mg/L

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

CONCLUSIONS

The inhalation L C 50 (1

- hr) was > 2.45 mg/L.

STUDY RELIABILITY

[3] Not reliable. Based on information provided in the summary , this study is considered 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 , which did not induce any mortalities, 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 f

ollowed.

REFERENCES

Thackar a , 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. Sponsor: Ethyl Corporation, Baton Rouge, LA. [ As summarized in Albemarle Corporation, 2011. ]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011) . Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes i s taken dir ectly from Albemarle Corporation (2011).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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20 Study 3: Acute dermal toxicity study in rabbits (Heenehan et al., 1977 b )

Title: Acute Dermal Toxicity Study

This study is: Adequate Not Adequate

This study is not considered to be adequate because an accurate LD 50 value could not be determined due to the lack of a clear dose

- response relationship . Additionally, i mportant study details were missing from the study summary including purity/composition of the test substance and test method/guideline followed.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; Clear, light colored liquid; the typical composition of the commercial product is 75

- 81% 3,5

- diethyltoluene

- 2,4

- diamine, 1 8

- 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 Toxicity

-- 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, 1410 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 throughoutFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

21 the 24 hr exposure period. Animals were observed for 14 d ays. ” [ Albemarle Corporation 2011, p. 11]

RESULTS

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

- bw, respectively

. Endpoint value(s): LD 50 = not determined

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

CONCLUSIONS

“ Due to inconsistent mortality, a meaningful LD 50 could not be determined. ” [ Albemarle Corporation 2011, p. 11]

STUDY RELIABILITY

[3] Not reliable. This study is not considered to be adequate because an accurate LD 50 value could not be determined due to the lack of a clear dose

- response relationship . Additionally, i mportant study details were missing from the study summary including purity/composition of the test subs tance and test method/guideline followed.

REFERENCES

Heenehan, P., Braun, W., and Rinehart, W. 1977 b . Acute Dermal Toxicity Study. Compound: Diethylated Toluenediamine. Project No. 4449

- 77. Performing Laboratory: Bio/dynamics Inc. S ponsor: Ethyl Corporation, Baton Rouge, LA. [As summarized in Albemarle Corporation, 2011.]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of Septem ber 12 , 2014.

Text in quotes is taken directly from Albemarle Corporation (2011).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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22 D

- 5 . Repeated

- Dose Toxicity

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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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 535 th mee ting on 12 May, 1981).

Test type: Subchronic 90

- Day Oral Repeated

- Dose Toxicity

-- Rat

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 and 320 ppm (0, 5, 12.5 and 32 mg/kg

- bw /day, respectively)Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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23 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. ” [Naismith and Mathews 1987, p. 4]

“ All raw data were summarized and statistically evaluated using LABCAT modules designed by Innovative Programming Associates, Inc., One Airport Place, Princeton, NJ 08540. ” [Naismith and Mathews 1987, p. 4]

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. ” [Naismith and Mathews 1987, p. 8] The concentration of DETDA in feed was measured analytically by gas chromatography approximately every two weeks.

“ 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 CO 2 asphyxiation. Any weak or moribund animals were removed, sacrificed and necropsied during the study as deemed necessary by the study director and principle investigator. ” [Naismith and Mathews 1987, pp. 8

- 9]

“ Blood was drawn by percutaneous cardiocentesis. All animal s were fasted overnight prior to the time of sacrifice. The following determinations [hematology and clinical chemistry] were made on twenty rats (10/sex) prior to initiation. ” [ Naismith and Mathews 1987, p. 9] A fter 90 days of feeding, 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, chlori de, 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 de tected all the animals would be examined. ” [Naismith and Mathews 1987, p. 9]

“ 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. ” [Naismith and Mathews 1987, p. 9] The following organs were weighed: liver, gonads, adrenal glands, brain and kidneys.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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24 “ The following organs and tissues from the animals at the 90

- day sacrifice were preserved in 10% neutral buffered for malin for future histopathological examination: all gross lesions, brain

including sections of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/ parathyroid, thymus, lungs, trachea, heart, sternum with bone marrow, salivary glands, l iver, 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 bladd er, 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. ” [Naismith and Mathews 1987, p. 10]

“ 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. ” [Naismith and Mathews 1987, pp. 10

- 11]

RESULTS

Endpoint value(s): LOAEL = 50 ppm (5 mg/kg

- bw /day; lowest dose tested) based on increased hematocrit in females, increased incidence and severity of multifocal degeneration of the acinar cells of the pancreas in males

, and increased severity of splenic pigmentation in females; NOAEL = Not established

.

Results remarks: Analytically measured concentrations of DETDA in feed ranged from 77 to 110% of target concentrations.

“ 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 t one, 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. ” [Albemarle Corporation 2011, p. 1 3 ]

“ Mean body weight of male and female rats in the high dose group (320 ppm) was [ stati sti cally ] 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) o n 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. ” [Albemarle Corporation 20 11, p. 1 3 ] “ 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,Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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25 the total gain for the high and mid dose groups w as significantly reduced. ” [Naismith and Mathews 1987, p. 1]

“ 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 reduc ed on Days 45, 59, 63, 73 and 90. Female food consumption in this dosage group w as 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. ” [Albemarle Corporation 2011, p. 1 3 ]

“ Twenty

- seven rats in the high dose group died during the study. Thirteen [ (4 m ales and 9 females) ] were found dead and fourteen [ (7 males and 7 females) ] were moribund sacrificed. The majority of the necropsy observations w as non

- specific, and included stomach lesions, dark discoloration of the liver, kidneys and/or genital organs and intestines, emaciation and apparent dehydration, lung congestion, liver foci and lack of thymic tissue. ” [Albemarle Corporation 2011, p. 1 3 ]

“ 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 r ecorded 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 co rtical 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. ” [Albemarle Corporation 2011, p. 13]

Evaluat ion of the blood chemistry parameters revealed statistically significant increases in SGPT, SGOT and BUN values in the high dose group (males and females)

. “ 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 exhi bited significant increase s in G G TP and decrease s in total protein content. ” [Albemarle Corp oration 2011, p. 14]

“ High dose d males exhibited statistically significant increases in erythrocyte and hemoglobin
counts. ” [Albemarle Corporation 2011, p. 14] H emoglobin content, leukocyte counts, platelet counts and mean corpuscular volume were significantly lower in high

- dose males than cont rol s. Hematocrit, hemoglobin, and erythrocyte values were statistically significantly higher in high

dose females relativ e to controls

. Low

- and mid

- dose females also exhibited significantly higher hematocrit values. Although increased hematocrit may be indicative of dehydration, drinking water intake was not assessed; therefore, the potential adverse nature of this effect cannot be dismissed.

“ Mean [ absolute ] 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. ” [AlbemarleFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

26 Corporation 2011, p. 14] Relative testes weight was also significantly increased in high

- dose males. “ In addition, the mid dose male and female liver, kidney and female brain weights to body weight we re 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.” [Albemarle Corporation 2011, p. 14]

“ 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 gast rointestinal tract with or without gas distention were observed in each treatment group . A few high dose rats, however, exhibited small spleens , little visible thymic tissue, and focal or linear stomach lesions. In general, the high dose animals appeared thin and dehydrated. ” [Albemarle Corporation 2011, p. 14]

“ 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 at 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 c hange), atrophy of many organs, lymphoid depletion of the spleen, thymus and mesenteric lymph node, and increased [severity of] pigmentation of the liver and spleen. ” [Albemarle Corporation 2011, p. 14] The incidence and severity of multifocal degeneratio n of the acinar cells of the pancreas w ere increased in males at 50 ppm and in males and females at 125 ppm of DETDA relative to controls

. The severity of splenic pigmentation (likely hemosiderin) in females receiving 50 ppm of DETDA in the diet w as also increased in
compar ison to controls

. Study authors did not identify the underlying pathophysiology of the splenic pigmentation; therefore, this effect is considered to be potentially adverse.

CONCLUSIONS

The LOAEL was 50 ppm ( 5 mg/kg

- bw /day ; the lowest dose tested ) based on increased hematocrit in females, increased incidence and severity of multifocal degeneration of the acinar cells of the pancreas in males, and increased severity of splenic pigmentation in females ; a NOAEL was not establis hed.

“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.” [Naismith and Mathews 1987, p. 15]

STUDY RELIABILITY

[ 2 ] Reliable with restrictions

. P urity/composition of the test substance was not provided.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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27 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). Perfo rming Laboratory: Pharmakon Research International, Inc. Waverly, PA. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes i s taken directly from Naismith and Mathews ( 1987 ) and Albemarle Corporation (2011)

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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28 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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 Che micals, ISBN 92

- 64

- 12221

- 4, adopted by the council at its 535 th meeting on 12 May, 1981. ” [Bolus and Mathews 1989, pp. 4

- 5]

Test type: Subchronic 28

- Day Oral Repeated

- Dose Toxicity

-- Rat

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 and 320 ppm (0, 5, 12.5, a nd 32 mg/kg

bw /day, respectively)Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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29 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 cont rol means. Tukey's test was used to determine significant differences between the DETDA lots at the same dose levels. ” [Bolus and Mathews 1989, p. 5]

“ All raw data were summarized and statistically evaluated using LABCAT modules designed by Innovative Pr ogramming Associates, Inc., One Airport Place, Princeton, NJ 08540 or SYSTAT, a PC based statistical package designed by SYSTAT, Inc,, Evanston, IL 60201. ” [Bolus and Mathews 1989, p. 5]

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 f or 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 i ntervals, 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. ” [Bolus and Mathews 1989, p. 1]

“ 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 cent ral 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. ” [Bolus and Mathews 1989, p p

. 9

- 10]

“ Blood was drawn by percutaneous cardiocentesis. All animals were fasted overnight prior to the time of sacrifice. The following determinations [hematology and clinical chemistry] 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, to tal 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 nit rogen, albumen, blood creatinine, total bilirubin, total serum protein measurements and serum insulin. ” [Bolus and Mathews 1989, p. 10]

“ Ophthalmological examinations using an ophthalmoscope were made prior to the administration of the test substance on D ays 14 and 28 of treatment and at the end of recovery (Day 56) on all animals. ” [Bolus and Mathews 1989, p. 10]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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30 “ All animals were subjected to necropsy, which included examination of the external surface of the body, all orifices, the cranial, thoracic, a bdominal and pelvic cavities and their contents. The following organs were weighed: liver, gonads, adrenal glands, brain and kidneys. ” [Bolus and Mathews 1989, p. 11]

“ The following organs and tissues from the animals sacrificed after 14 and 28 days of tr eatment 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, 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 turbinat es, 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, midtho racic and lumbar) and exorbital lachrymal glands. ” [Bolus and Mathews 1989, pp. 11

- 12]

“ 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 )

. ” [Bolus and Mathews 1989, p 12]

“ 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. ” [Bolus and Mathews 1989, p 12]

RESULTS

Endpoint value(s): NOAEL (Run 544, Drum 6131) = 50 ppm (5 mg/kg

- bw/day); LOAEL (Run 544, Drum 6131) = 125 ppm (12.5 mg/kg

- bw/day)

, based on increased incidence and severity of compound

- related degenerative changes in the acinar pancreas

.
NOAEL (Lot 2

- 87) = 50 ppm (5 mg/kg

- bw/day); LOAEL (Lot 2

- 87) = 125 ppm ( 12. 5 mg/kg

bw/day)

, based on increased incidence and severity of compound

- related degenerative changes in the acinar pancreas

.

Results remarks: Analytical measurements of DETDA in f eed ranged from 94 to 107% of target feed concentrations for Run 544 and 87 to 108% for Lot 2

- 87.

“ 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, decreasedFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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31 body tone, piloerection and excess ive defecation and scab formation. ” [Albemarle Corporation 2011, p. 15]

“ No rats died on test prior to scheduled sacrifice. ” [Albemarle Corporation 2011, p. 15]

“ Mean male body weights in the high dose groups were statistically lower than the control mea n 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. ” [Albemarle Corporation 2011, p. 15] Female body weights were significantly reduced in both high dose groups compared to the control mean on Day 14. Other than a significant reduction in body weight in high

- dose females of Lot 2

- 87 on Day 42, body weights of treated females (both lots) were comparable to controls throughout the recovery period.

“ 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 n oted for the females. ” [Albemarle Corporation 2011, p. 15]

D aily body weight gains were significantly reduced at several intervals for the males in the Run 544 125 and 320 ppm groups and the Lot 2

- 87 320 ppm group when compared to the controls. Female body weight gains in both the high dose groups were less severely affected ; however, significance was indicated when compared to the controls on Day 14 and Day 28

. Statistically significant differences were also noted when the daily gains of the different DETDA treatment groups were compared. H igh

- dose males of the Run 544 group showed decreased daily body weight gain on Day 56 when compared to high

- dose males of the Lot 2

- 87 group

. The total mean daily gain was also significantly reduced in the high dose Run 544 group compared to the high

- dose Lot 2

- 87 group

. There were no such differences observed for the females.

“ The daily food consumption for males was significantly reduced in both high dose DETDA groups at several time periods [ during treatment (but not recovery) ] , 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. ” [Albemarle Corporati on 2011, p. 15]

“ 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 oc ular 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. ” [Albemarle Corporation 2011, pp. 15

- 16]

“ The majority of necropsy observations recorded at either 14 or 28 [ day ] interim sacrifices w ere

non

- specific in nature [and low in incidence and not dose

- related]

. These observations included enlarged mandibular lymph nodes, scabs on the skin, lung and li ver 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 [po st

- recovery] ) included similar observations of the lungs, livers and kidneys ” [Albemarle Corporation 2011, p. 16]

. In addition, changes which were specific toFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

32 the high dose Run 544 male group included reduced adipose tissue, rough hair coat, small salivary glands, spleens, testes, seminal vesicles, epididymides and prostate; dark and small pancreas, and gastrointestinal tract lesions. “ There were no similar DETDA related changes noted at necropsy for the females

. ” [Albemarle Corporation 2011, p. 16 ]

At Day 14, no statistically significant differences were observed in the absolute organ weights of dosed groups (males or females) compared with controls

, but s tatistically significant increase s

were seen in relative [to body weight] brain and testes weights in high

- dose Lot 2

- 87 males

. High

- dose males of the Run 544 group also had relative [to body weight] testes weights that

were significantly higher than controls

. There were no significant changes in relative [to body weight] organ weights in fema les at Day 14 when the dosage groups were compared to the controls. At Day 14, no significant differences in relative [to brain weight] organ weights were observed at any dose of the DETDA materials in either sex, compared with controls. When comparisons were made at Day 14 between groups treated with Lot 2

- 87 or Run 544 at the same dose level, no significant differences were observed for relative [to body weight or to brain weight ] organ weights

. At Day 28, no statistically significant differences were observed in absolute organ weights of dosed groups (males or females) compared with controls

, but male relative [to body weight] liver weights and female relative [to body weight] kidney weights were significantly increased in high

- dose groups treated with Lot 2

- 87 or Run 544, compar ed with control. Relative [to body weight] brain and liver weights were also significantly increased at Day 28, compared with control, in females treated with Run 544. A t Day 28, no significant differences in relative [to brain weight] organ weights were observed at any dose of the DETDA materials in either sex, compared with controls. When comparisons were made between groups treated with Lot 2

- 87 or Run 544 at the same dose level, no significant differences were observed for relative [to body weight or to brain weight ] organ weights at Day 28.

At the Day 56 terminal necropsy, significantly decreased absolute liver and absolute testes weights were observed in high

- dos e males of the Run 544 group (but not the 2

- 87 group) compared to the control group. Comparisons by dosage group between the two DETDA materials indicated the high dose male absolute liver and absolute testes weights in the Run 544 high

- dose group were si gnificantly lower than the Lot 2

- 87 high

- dose group at the terminal (Day 56) necropsy. At Day 56, significant relative [to body weight] weight increases, compared with control, were seen for male brain, kidneys, and adrenal glands and female liver at the high dose of Run 544 and for female kidneys at the high dose of Lot 2

- 87. When comparisons were made between male groups treated with the two DETDA material s at the same dose level, relative [to body weight] brain and kidney weights were significa ntly higher in the Run 544 high

- dose males than the Lot 2

- 87 high

- dose males at Day 56. When comparisons were made at Day 56 between female groups treated with Lot 2

- 87 or Run 544 at the same dose level, no significant differences were observed for relati ve [to body weight] organ weights

. At Day 56, relative [to brain weight] liver weight was significantly decreased in high

- dose Run 544 males compared with control. When comparisons were made between groups treated with Lot 2

- 87 or Run 544 at the same dos e level, no significant differences were observed for relative [to brain weight] organ weights in either sex at Day 56.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

33 The significant increase in relative [to body weight] kidney weight that was seen in high

- dose males of the Run 544 group at the 56

- day sacrifice appears to correlate with vacuolation of the tubular cells of the kidney that was present in two of five of th e males of this group. Otherwise,

a lthough numerous significant changes were seen at high dose in absolute or relative weights of the brain, liver, adrenals, or testes compared with control, these changes were not accompanied by histopathological changes and

, therefore, are unlikely to be toxicologically significant.

“ 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. ” [Albemarle Corporation 2011, p. 17]

Low

- dose (but not mid

- or high

- dose) Lot 2

- 87 male platele t counts were significantly higher than the controls at the Day 14 interim necropsy. At Day 28 necropsy , Run 544 high

- dose male s and mid

- dose female s showed platelet counts that were significant ly increased compared to controls. Increases in platelets observed at 14 days in low

- dose males (Lot 2

- 87) and at 28 days in high

- dose males and mid

- dose males are not likely related to treatment, as effects do not exhibit consistent dose

- or duration

- dependence. 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 in comparison to controls.

Comparisons of DETDA materials at the Day 28 interim sacrifices indicated the platelet counts for Run 544 DETDA treated female s were significantly higher than the Lot 2

- 87 females.

When comparisons were made between DETDA formulations for the Day 56 necropsy , the platelet and leucocyte counts were significantly lower in the high dose Run 544 males com pared to the high dose Lot 2

- 87 males

.

Multifoc a l d egenerative changes in the acinar cells of the pancreas were detected in DETDA

treated rats at 125 and 320 ppm (males) or 320 ppm (females) ; similar changes were not observed at the 50 ppm dose of either lot or sex. The severity of these lesions increased with increasing dose. Male rats were affected at an earlier time and more severely than the female rats. At the Day 28 sacrifice, acinar degeneration was accompanied by diffuse acinar atrophy in all 2

- 87 and Run 544 males and females at 320 ppm. The 28

- day recovery period allowed for an almost complete recovery from the pancreatic effects of DETDA Lot 2

- 87 with decreased severity of multifocal acinar degeneration and decreased incidence and severity of diffuse acinar atrophy. One Lot 2

- 87 male had moderate vacuolation of the islet cells at 320 ppm

, while three of five males exposed to 320 ppm DETDA Run 544 Drum 6131 showed moderate to moderately

severe islet cell vacuolation and secondary changes of cachexia and diabetes. At the day 56 sacrifice (recovery group), vacuolation of the tubular cells of the kidney was seen in two of five of the males that were exposed to 320 ppm DETDA Run 544 Drum 6131; as noted above, this
appea rs to correlate with a significant increase in relative [to body weight] kidney weight in males at this dose , compared with control

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

34 “ In summary, the results of this study should be evaluated with consideration for those of the previous 90

- d study. The r esults 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 c ataracts 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 to xicity between lots was observed. After exposure for 28 consecutive days, DETDA Lot 2

- 87 exerted an effect, but was less toxic than the Run 544, Drum 6131 preparation. ” [Albemarle Corporation 2011, p. 18]

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. ” [Bolus and Mathews 1989, p. 24]

The N OAEL and L OAEL values for Run 544, Drum 6131 and Lot 2

- 87 were 5 0 and 12 5 ppm (5 and 12. 5 mg/kg

- bw /day) , respectively, based on increased incidence and severity of compound

related degenerative changes in the acinar pancreas.

STUDY RELIABILITY

[ 2 ] Reliable with restrictions. P urity/composition of the test substance was not provided.

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 Corporat ion, Baton Rouge, LA.

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission availab le online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes i s taken directly from Bolus and Mathews ( 1989 ) and Albemarle (2011)

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

35 Study 3: 28

- Day repeated

- dose oral toxicity study in rats ( Naismith and Matthews , 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 an d histopathology analyses were not performed. Additionally, the purity and composition of the test substance were not provided

.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; Physical state: Amber

- colored liquid; Lot: Run #544, Drum # 6131

METHOD

Method/guideline followed: Not indicated

Test type: Subchronic 28

- Day Oral Repeated

- Dose Toxicity

-- Rat

GLP compliant?: Yes

Year study performed: 1986

Test species: Sprague

- Dawley rats from Charles River Laboratories, Wilmington, Massachusetts; Rats used in this study were 41 days old and weighed 156

- 195 g (males) and 114

- 135 g (females) at initiation.

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 and 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 diff erences among the means were indicated, Dunnett’s test was used to determine significant differences from control means (p ≤ 0.05). ” [Naismith and Matthews 1986, p. 3]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

36 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 toxicit y. 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. FD A, U.S. EPA, and OECD Good Laboratory Practices. ” [Albemarle Corporation 2011, p. 11]

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 d ose 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 g roup were sacrificed on day 17. ” [Albemarle Corporation 2011, p. 11]

“ 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. ” [Albemarle Corporation 2011, pp. 11

- 12]

“ 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 leve l. 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. ” [Albemarle Corporation 2011, p. 12]

“ 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

- relatedFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

37 lesions recorded at terminal necropsy in any of the treated animals. ” [Albemarle Corporation 2011, p. 12]

“ 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 in the mean relative kidney weights at the 1200 ppm level. No s tatistically significant differences for relative organ weights were observed in the female rats. ” [Albemarle Corporation 2011, p. 12]

CONCLUSIONS

The LOAEL was 400 ppm (40 mg/kg

- bw/day) in males and females, based on reduced food consumption in both se xes and reduced body weight in females. The NOAEL was 40 ppm (4 mg/kg

- bw/day).

STUDY RELIABILITY

[3] Not reliable. In this range

- finding study, clinical chemistry, hematology and histopathology analyses were not performed. Additionally, the purity and composition of the test substance

were not provided

.

REFERENCES

Naismith, R.

, and Matthews, 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.

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes i s taken directly from Naismith and Matthews ( 19 86 ) and Albemarle Corporation (2011)

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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38 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: Benzenediamine, ar,ar

- diethyl

- ar

- methyl, CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA); Referred to as “diethyltoluamine”

, “ DETA ”, and “diethyltoluenediamine” in the study report.

Remarks: Purity: 95.78%; Brown liquid; Isomer Ratio: 80:20 2,4

- DETDA:2,6

- DETDA

METHOD

Method/guideline followed: Not indicated

Test type: Subchronic 21

- Day Dermal Repeated

- Dose Toxicity

-- Rabbit

GLP compliant?: Not indicated

Year study performed: 1980

Test species: New Zealand White rabbits ; obtained from Small Stock, Inc., Pea Ridge, Arkansas; rabbits weighed 2.31

- 3.59 kg (males) and 2.40

- 3.17 kg (females) and were ~ 14 wks at study initiation

.

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, and 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. ” [Lamb et al. 1981, p. 8]

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 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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39 “ 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 th e 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 o f 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. ” [Lamb et al. 1981 , pp. 6

- 7]

“ 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 m ethod 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. ” [Lamb et al. 1981, p. 7]

“ 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 laborato ry testing both before initiating the study and on the day of the last treatment. ” [Lamb et al. 1981, p. 7]

“ Hematology tests included determination s of hematocrit and hemoglobin concentration, red and white blood cell counts, differential white cell coun ts and platelet counts. ” [Lamb et al. 1981, p. 7]

“ 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. ” [Lamb et al. 1981, p. 7]

“ All test and control animals were subjected to gross necropsy. The liver, kidne ys, heart, gonads, thyroid (with parathyroid), adrenals, lungs, brain and pituitary were weighed. Tissues were collected and fixed in 10% buffered formalin for histopathology examination. ” [Lamb et al. 1981, p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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40 RESULTS

Endpoint value(s): LOAEL (systemic toxicity) > 100 mg/kg

- bw/day

, based on no significant effects at the highest dose tested; NOAEL = 100 mg/kg

- bw/day.

LOAEL (application site irritation) = 10 mg/kg

- bw/day

, based on mild to moderate irritation; NOAEL = 1 mg/kg

- bw/day

.

R esults remarks: “ On the first day of treatment, it was observed that a male rabbit to be treated with 100 mg/kg (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 treatment, its condition could not have been related to treatment. No mortality occurred in treated rabbits. ” [Lamb et al. 1981, p. 9]

“ No significant differences were apparent among mean body weights of either sex throughout the study. However, two female rabbits receiving 100 mg/kg and one receiving 10 mg/kg failed to gain weight throughout the study. All of these showed initial weight gain followed by loss. Of the two rabbits rec eiving 100 mg/kg, 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 exhibited only approximately half of the total average weight gain of her group. No dif ferences in rate of weight gain were apparent among male rabbits treated at this dosage. ” [Lamb et al. 1981, p. 9]

“ 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. ” [Lamb et al. 1981, p. 9]

“ No evidence of skin irritation was observed in control rabbits of either sex. Slight erythema accompanied occasionally by slight edema wa s 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 t reated 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 we re 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. ” [Lamb et al. 1981, p. 9]

“ 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. ” [Lamb et al. 1981, p. 10] For the results after treatment

, “ occasional differences in hematology values w ere observed among groups of male rabbits, but these were not outside the range of normal biological v ariation . In female rabbits, a dose

- related decrease in monocyte counts was observed; the counts were significantly different from control females forFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

41 th ose receiving 10 and 100 mg/kg DETA but not for those receiving 1.0 mg/kg. However, the counts in these two groups were within the range of counts (0

- 11) observed in rabbits of the same sex and appro ximate age. No other differences of biologic significan ce were observed in female rabbits. ” [Lamb et al. 1981, p. 10]

“ 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 DETA and in males rece iving 100 mg/kg. The sodium concentrations were statistically significantly different from control in all treated groups of females and in males receiving 10 mg/kg DETA but not those receiving 100 mg/kg. Decreased serum potassium concentrations were obse rved 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 physiologic processes. No other differences in chemistry results in male or female rabbits were observed that were outside the range of normal biological variation. ” [Lamb et al. 1981, p. 10]

“ 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 DETA. 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 r esult of applications of DETA. ” [Lamb et al. 1981, p. 11]

“ 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. ” [Lamb et al. 1981, p. 10]

CONCLUSIONS

The N OAEL for systemic toxicity is 100 mg/kg

- bw/day, based on no adverse eff ects at the highest dose tested

.

The LOAEL for application site irritation is 10 mg/kg

- bw/day

, based on mild to moderate irritation; the NOAEL is 1 mg/kg

- bw/day

.

“ Repeated dermal application of diethyltoluamine at 1.0 mg/kg resulted in very slight irritation of no biologic significance. Application of 10 and 100 mg/kg resulted in mild to moderate local irritation and open wounds were observed in skin of some rabbits treated at 100 mg/kg. No other significant overt toxicity was observed in treated rabbits. ” [Lamb et al. 1981, p. 12]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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42 “ 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. ” [Lamb et al. 1981, p. 12]

“ 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. ” [Lamb et al. 1981, p. 12]

“ Repeated application of DETA at 1.0, 10 or 100 mg/kg resulted in no significant systemic effects. Application of 10 and 100 mg/kg resulted in si gnificant local effects on treated skin; these effects were more pronounced at the highest dosage. No significant local effects were observed after application of DETA at 1.0 mg/kg. ” [Lamb et al. 1981, p. 12]

STUDY RELIABILITY

[2] Reliable with restrict ions. T est method/guideline followed and GLP compliance w ere not specified.

REFERENCE S

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.

Text in quotes i s taken directly from Lamb et al. ( 1981 )

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

43 D

- 6 . Genetic Toxicity

– Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity studies in Salmonella typhimurium (Barfknecht et al. , 1986 a )

Title: Ames Salmonella /microsome plate test

This study is : Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as DETDA

- OBG in the study report

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 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, dose

related 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 (1983)

. This program applies a linear regression analysis to the data points and any P value greater than 0.0 5 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 assa y. Alternatively, if the solvent control is within the 95% confidence limits of the historical mean (See Historical Data) for control valuesFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

44 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. ” [Barfknecht et al. 1986 a , p. 6]

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. ” [Barfknecht et al. 1986 a , p. 3] Positive controls not requiring metabolic activation were strain specific and included the follow ing : 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 posi tive control requiring metabolic activation was 2

- anthramine (2

- A minoanthracene) (2.5 μg/plate)

... in all strains. ” [Barfknecht et al. 1986 a , p. 4]

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 Isotemp R 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 ab sence of a confluent lawn of bacteria. ” [Barfknecht et al. 1986 a , p. 4]

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, an d maintained atFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

45 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp R 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 tr iplicate. 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 po ured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. ” [Barfknecht et al. 1986 a , p. 5]

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. ” [Barfknecht et al. 1986 a , p. 5]

“ 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. ” [Barfknecht et al. 1986 a , p. 5]

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 0.5 mM histidine

- 0.5 mM 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. ” [Barfknecht et al. 1986 a , p. 6]

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. ” [Barfknecht et al. 1986 a , p. 6]

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. ” [Barfknecht et al. 1986 a , p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

46 “ The test article induced a statistically significant (at the 95% confidence limit) linear mutational dose resp onse 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. ” [Barfknecht et al. 1986 a , pp. 7

- 8]

“ 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 confirmat ory 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 6 0 00 μg/plate dose level in the second mutation assay. ” [Barfknecht et al. 1986 a , p. 8]

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 va lues that were within the acceptable limits of mean historical data. ” [Barfknecht et al. 1986 a , p. 8]

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. ” [Barfknecht et al. 1986 a , p. 8]

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCES

Barfknecht, T., Naismith, R. , and Matthews, R. 1986 a . Ames Salmonella /Microsome Plate Test with DETDA

- OBG 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 Rou ge, LA.

Text in quotes is taken directly from Barfknecht et al. (1986 a ).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

47 Study 2: Mutagenicity studies in Salmonella typhimurium ( Barfknecht et al., 1986

b )

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

This study is : Adequate Not Adequate

TEST SUBSTANCE

Identity: 3,5

- Diethyltoluene

- 2,6

- diamine, CASRN 2095

- 01

- 4 ( 2,6

- DETDA isomer of CASRN 68479

- 98

- 1) ; Synonym: 2,6

- DETDA ; Referred to as 2,6 DETDA #2 in the study report

Remarks: 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 μ g/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% confi dence l imit 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 hig her 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 limi ts 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 a s the absence of a reproducible increase in the number of histidine

- independent colonies. ” [ Barfknecht et al . 1986 b , p. 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

48 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. ” [ Barfknecht et al . 1986 b , p. 3] Positive controls not requiring metabolic activation were strain specific and included the follow ing :

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. ” [ Barfknecht et al . 1986b, p. 4]

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 supplement ed 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 Isotemp R Dry Bath at 45°C. All control and treated tubes and plates were don e 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 pla tes 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 confluen t lawn of bacteria. ” [ Barfknecht et al . 1986 b , p. 4]

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 Fis her lsotemp R Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. AllFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

49 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. ” [ Barfknecht et al . 1986 b , p. 5]

Metabolic Activation System: “ Tubes requiring metabolic activation had, in addition to the preceding top agar ingredi ents, an S

- 9 fraction of rat liver homogenate obtained from Aroclor 1254

- treated Sprague Dawley rats. ” [ Barfknecht et al . 1986 b , p. 5]

“ 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

. W ithin an hour the plates were i nverted 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

. ” [ Barfknecht et al . 1986 b , p. 5]

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. ” [ Barfknecht et al . 1986 b , p. 6]

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. ” [ Barfknecht et al . 1986 b , p. 6]

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. ” [ Barfknecht et al . 1986 b , p. 7]

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. ” [ Barfknecht et al . 1986 b , p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

50 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

Barfknecht , T., Naismith, R., and Matthews, R. 1986 b . 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 Laborat ories, Scranton, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Barfknecht et al . (1986 b ).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

51 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Lot # 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 / M icrosome 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 si gnificant. 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 chemica l is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine

- independent colonies. ” [ Barfknecht et al . 1985a, p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

52 Test method/conditions remarks: Negative and Positive Controls: “ 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 t he test system, all tester strains were also run with known positive response chemicals, six (6) plates being seeded per point. ” [ Barfknecht et al . 1985a, p. 3]

“ The positive control requiring metabolic activation was 2

- anthramine (2

- Aminoanthracene) (2.5 μg/plate)

... in all strains. ” [ Barfknecht et al . 1985a, p. 3]

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. ” [ Barfknecht et al . 1985a, p. 1]

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 Isotemp R Dry Bath at 45°C

... [ All negative and po sitive 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 t ester 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. ” [ Barfknecht et al . 1985a, p. 4]

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. ” [ Barfknecht et al

. 1985a, p. 4]

“ 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

. W ithin an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for approximately 48 and 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfa ced 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. ” [ Barfknecht et al

. 1985a, pp. 4

- 5]

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 t op 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 mixFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

53 were added to 2 ml of molten top agar supplemented with 0.5mM histidine

- 0.5mM biotin and poured onto minimal glucose plates. To p 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. ” [ Barfknecht et al . 1985a, p. 5]

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. ” [ Barfknecht et al . 1985a, p. 5]

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

- 1 0,000 μg/plate at approximately 4 8 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, respec tively. 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. ” [ Barfknecht et al

. 1985a, pp. 6

- 7]

Genotoxicity result: Positive with metabolic activation

Cytotoxic concentration (if applicable): N ot 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 limits of mean historical data except the 72 hour incubation solvent control value for strain TA98. The sponso r requested that the laboratory not repeat strain TA98. ” [ Barfknecht et al . 1985a, p. 7]

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 report , 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

54 Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Barfknecht et al . (1985a).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

55 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: 3,5

- Diethyltoluene

- 2,4

- diamine, CASRN 2095

- 02

- 5 ( 2, 4

- DETDA isomer of CASRN 68479

- 98

- 1 ) ; Referred to as 2,4

- DETDA in the study report.

Remarks: Lot No. 5712

- 38

- 7; Clear yellow liquid; Composition: 99.953% 3,5

- diethyltoluene

2,4

- 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 solv ent 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 conside red positive. A negative result is defined as the absence of a reproducible increase in the number of histidine

- independent colonies. ” [ Barfknecht et al . 1985b, pp. 5

- 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

56 Test method/conditions remarks: Negative and Positive Controls: “ Tester strains TA 1535, 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. ” [ Barfknecht et al . 1985b, p. 3] Positive controls not requiring metabolic activation were strain specific and included the following :

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. ” [ Barfknecht et al . 1985b, p. 4]

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 Pl ate Incorporation Mutation Assay in consultation with the sponsor were 625, 1250, 2500, 5000 and 7500 μg/plate. ” [ Barfknecht et al . 1985b, p. 1]

“ The test article, 2,4

- DETDA, was evaluated in Ames/ Salmonella tester strains TA1535, T A 1537, 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. ” [ Barfknecht et al . 1985b, p. 1]

Top Agar: “ Top agar, used as a n 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 lsotemp R 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 ali quot 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. ” [ Barfknecht et al . 1985b, p. 4]

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. ” [ Barfknecht et al . 1985b, p. 5]

“ 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

. W ithin an hour the plates were inverted and placed in a dark 37°C incubator. The platesFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

57 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. ” [ Barfknecht et al . 1985b, p. 5]

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 volum es as in the assay

. The test substance or S

- 9 mix were added to 2 ml of molten top agar supplemented with 0.5 m M 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. ” [ Barfknecht et al . 1985b, p. 5]

Data Reporting: “ In scoring the assay, the positive and negative controls were first evaluated. If the negative co ntrol values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. ” [ Barfknecht et al . 1985b, p. 5]

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. ” [ Barfknecht et al

. 1985b, p. 7]

“ Mutant frequency values that were 2

- fold the mean solvent control mutant frequency value 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 observati on or the occurrence of a dose response at lower concentrations could not be confirmed. ” [ Barfknecht et al . 1985b, p. 7]

Genotoxicity result: Equivocal

Cytotoxic concentration (if applicable): N ot 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 values. ” [ Barfknecht et al . 1985b, p. 7]

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 strai n 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 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

58 STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study report 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

- 013

- 85. Performing Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl Corporation.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corpo ration, Baton Rouge, LA.

Text in quotes is taken directly from Barfknecht et al . (1985b).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

59 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: 3,5

- Diethyltoluene

- 2,6

- diamine, CASRN 2095

- 01

- 4 ( 2,6

- DETDA isomer of CASRN 68479

- 98

- 1 ) ; Referred to as 2,6

- DETDA in the study report.

Remarks: Lot No. 5712

- 53

- 73; Light brown solid; Composition: 99.51% 3,5

- diethyltoluene

2,6

- 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 / M icrosome 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 l imit 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 sol vent 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 consid ered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine

- independent colonies. ” [ Barfknecht et al . 1985c, pp. 5

- 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

60 Test method/conditions remarks: Negative and Positive Controls: “ Tester strains TA 1535, 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 t he integrity of the test system, all tester strains were also run, in triplicate, with known positive response chemicals. ” [ Barfknecht et al . 1985c, p. 3] Positive controls not requiring metabolic activation were strain specific and included the following :

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. ” [ Barfknecht et al . 1985c, p. 4]

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. ” [ Barfknecht et al . 1985c, p. 1]

“ The test article, 2,6

- DETDA, was evaluated in Ames/ Salmonella tester strains TA1535, TA1537, TA1538, TA98 a nd TA100 with and without metabolic activation preparation. There was 0.10 ml S

- 9 supernatant (45 mg protein per ml) per 1.0 ml of S

- 9 mix in the rat liver metabolic activation preparation. ” [ Barfknecht et al . 1985c, p. 1]

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 lsotemp R 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 t op agar overlay was allowed to harden. ” [ Barfknecht et al . 1985c, p. 4]

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. ” [ Barfknecht et al . 1985c, p. 5]

“ 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

. W ithin an hour the plates were inverted and placed in a dark 37°C incubator. The platesFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

61 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. ” [ Barfknecht et al . 1985c, p. 5]

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 volu mes as in the assay

. The test substance or S

- 9 mix were added to 2 ml of molten top agar supplemented with 0.5 m M 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. ” [ Barfknecht et al . 1985c, p. 5]

Data Reporting: “ In scoring the assay, the positive and negative controls were first evaluated. If the negative co ntrol values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. ” [ Barfknecht et al . 1985c, p. 5]

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 s train TA1535 with metabolic activation preparation over the same dose range. Statistic ally 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. ” [ Barfknecht et al . 1985c, p. 7]

Genotoxicity result: Positive with metabolic activation

Cytotoxic concentration (if applicable): N ot 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. ” [ Barfknecht et al . 1985c, p. 7]

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 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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62 STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study report and included cytotoxicity of the test substance.

REFERENCES

Barfknecht, T., Naismith, R., and Matthews, R. 1985c. Ames Salmonella /Microsome Plate Test with 2,6 D ETDA 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 Corpo ration, Baton Rouge, LA.

Text in quotes is taken directly from Barfknecht et al . (1985c).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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63 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA); Referred to as Ethacure 100 in the study report.

Remarks: Purity not specified; 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

Statisti cal 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 TA 1535, 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. ” [Stewart and Riccio 1985, p. 9]

“ After consultation with the clien t, the preliminary (range

- finding) a ssay was waived because this sample i s 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. ” [Stewart and Riccio 1985, p. 9]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

64 Metabolic Activation: Aroclor 1254

- induced, rat

- liver S

- 9; SRI Batch F

- 6; ~ 31.9 mg/ml protein; Animal Supplier: Simonsen Laboratories, Gilroy, CA

Negative (Diluent) Control Material: Dimethylsulfoxide (DMSO)

Positive Control Chem icals: 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. ” [Stewart and Riccio 1985, pp. 7

- 8]

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. ” [Stewart and Riccio 1985, p. 8]

Inconclusive: “ When a test article cannot be identified clearly as a mutagen or nonmutagen in t he standard plate assay, the results are classified as inconclusive. ” [Stewart and Riccio 1985, p. 8]

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 histid ine, 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 presen ce of metabolic activation. Toxicity

-- indicated by a decrease in the number of revertants with or without concurrent thinning of the background growth la wn

- - was seen at the highest dose level with several of the tester strains. ” [Stewart and Riccio 1985, p. 9]

“ 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 lev els. 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. ” [Stewart and Riccio 1985, pp. 9

- 10]

Genotoxicity result: Positive with metabolic activation

Cytotoxic concentration (if applicable): 5000 μg/plateFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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65 Results remarks: “ Positive and negative (diluent) control values were within acceptable limits for all assays. ” [Stewart and Riccio 1985, p. 9]

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

w as not mutagenic in S. typhimurium strains TA1535 or TA1537 with or without metabolic activation.

STUDY RELIABILITY

[2] Reliable with restri ctions. Important study details were missing from the study report 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.

Text in quotes is taken directly from Stewart and Riccio (1985).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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66 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Clear brown liquid; Lot #5868

- 1 ; 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

- 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 me tabolic 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. ” [Johnson and Pettigrew 1987a, p. 4]

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. ” [Johnson and Pettigrew 1987a, p. 4]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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67 Genotoxicity result: Positive with metabolic activation

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. ” [Johnson and Pettigrew 1987a, p. 4]

CONCLUSIONS

“ DETDA was genetically active in the Salmonella / Microsomal Assay according to Protocol

- Ettox 027. ” [Johnson and Pettigrew 1987a, p. 4]

STUDY RELIABILITY

[2] Reliable with restrictions. T he summary did not indicate whether or not the solvent and positive controls responded appropriately

.

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.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Johnson and Pettigrew (1987a).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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68 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; R eferred to as “DETA” and identified as “diethyltoluenediamine” in the study report.

Remarks: Purity not specified; Dark amber liquid

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 empl oyi ng Salmonella and Saccharomyces indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from human liver. ” [Jagannath and Brusick 1977, p. 5]

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/pl ate) 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).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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69 “ 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. ” [Jagannat h and Brusick 1977, p. 2]

Plate Test (Overlay Method): “ Approximately 10 8 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 chem ical 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 an d those that require metabolic activation were run with each assay. ” [Jagannath and Brusick 1977, p. 3]

“ 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 provi ded on t he computer printout. ” [Jagannath and Brusick 1977, p. 3]

RESULTS

“ The compound was tested over a series of concentrations such that there was either quantitative

or quali tative 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 t o 5 μl per pla te. The compound was slightly toxic to the strain TA

- 1535 and TA

- 1537 at 5 μl per p l ate. ” [Jagannath and Brusick 1977, p. 5]

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. ” [Jagannath and Brusick 1977, p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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70 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 whether or not the solvent and positive controls responded appropriately

.

REFERENCES

Jagannath, D., and Brusick, D. 1977. Mutagenic ity Evaluation of DETA (Diethyltoluenediamine) Final Report. Performing Laboratory: Litton Bionetics, Kensington, MD. LBI Project No. 20838. Sponsor: Ethyl Corporation.

Text in quotes is taken directly from Jagannath and Brusick (1977).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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71 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

- , CASRN 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 / M icrosome 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. Significanc e at the 95% confidence l imit 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 th e 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 9 5% 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 r esult is defined as the absence of a reproducible increase in the number of histidine

- independent colonies. ” [Barfknecht et al . 1985d, p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

72 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 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 0.0 8 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. ” [Barfknecht et al . 1985d, p. 1]

“ 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. ” [Barfknecht et al . 1985d, p. 3]

“ The positive control requiring metabolic activation was 2

- anthramine (2

- Aminoanthracene) (2.5 μg/plate)

... in all strains. ” [Barfknecht et al . 1985d, p. 3]

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 I sotemp R Dry Bath at 45°C

... [ All negative and pos itive 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 s of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound and a 0.5 ml of S

- 9 metabolic activation preparation

. 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. ” [Barfknecht et al . 1985d, p. 4]

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 harde n for several minutes. The same procedure was repeated for each tester strain

. W ithin an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for approximately 48 and 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 c olony morphology. ” [Barfknecht et al

. 1985d, p. 4]

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, solv ent, top agar and S

- 9 mix at the same volumes as in the assay

. The test substance or S

- 9 mixFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

73 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. ” [Barfknecht et al . 1985d, p. 5]

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. ” [Barfknecht et al . 1985d, p. 5]

RESULTS

“ The results for test article 57 11

- 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. I n addition, maximum fold increase mutant frequency values which were observed at the 48 hour inc ubation period were 19.7, 22.8 and 3.4 for strains TA1538, TA98 and TA100, respectively. ” [Barfknecht et al . 1985d, p. 1]

Genotoxicity result: Positive with metabolic activation

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 a fter the 72 hour incubation period. The sponsor requested that the laboratory not repeat strain TA98. ” [Barfknecht et al . 1985d, p. 1]

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. ” [Barfknecht et al . 1985d, p. 7]

STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study summary provided and included test metho d/guidelines followed and cytotoxicity of the 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

74 Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Barfknecht et al . (1985d).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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75 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified ; 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 so lvent 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 artic le 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 increa se in the number of histidine

- independent revertants (or three

- fold increase in the absence of any dose

- dependency). ” [ Stankowski and Naismith 1987a, p. 4]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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76 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 homo genate with the appropriate buffer and cofactors. ” [ Stankowski and Naismith 1987a, p. 1]

“ 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 as says 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, 1 0,000 and 12,500 μg/plate in the presence of S 9. 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, 3750, 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, 25 0, 500, 1000, 2500, 3750 and 5000 μg/plate with S9. ” [ Stankowski and Naismith 1987a, p. 3]

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 MgCl 2 , 33mM KC l , 4mM NADP, 5mM glucose

- 6

- phosphate, 100mM NaH 2 PO 4 (pH 7.4) and 6% (v/v) Aroclor 1254

- indu ced male Sprague

- Dawley rat liver homogenate. The tubes were vortexed and 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 ho urs. ” [ Stankowski and Naismith 1987a, p. 4]

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 pl ating was done in triplicate. Plates were incubated for 48 hours at 37°C and then scored for bacterial growth. ” [ Stankowski and Naismith 1987a, p. 4]

Scoring: “ Following the 48

- hour incubation, the background lawn and spontaneous revertants were scored f

or 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 coloniesFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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77 were enumerated on an Artek electronic colony counter interfaced with an Apple c omputer 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 ...” [ Stankowski and Naismith 1987a, p. 4]

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 appr oximately 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

- 75 0 0 μg/plate; higher doses produced lower revertant freq uencies due to toxicity. ” [ Stankowski and Naismith 1987a, p. 1]

“ 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 st rains 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 wit h 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. ” [ Stankowski and Naismith 1987a, p. 1]

Genotoxicity result: Positive w ith metabolic activation

Cytotoxic concentration (if applicable): ≥ 7500 μg/plate

Results remarks: “ All positive and negative control values in both assays were within acceptable limits. ” [ Stankowski and Naismith 1987a, p. 1]

CONCLUSIONS

T herefore, “t he 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. ” [ Stankowski and Naismith 1987a, p. 1]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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78 STUDY RELIABILITY

[2] Reliable with restrictions. Important study details were missing from the study provided and included purity and composition of the test substance.

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.

Text in quotes is taken directly from Stankowski and Naismith (1987a).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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79 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as “DETA” and identified as “diethyltoluenediamine” in the study report.

Remarks: Purity not specified; 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: 1. 2 ml molten agar (0.6% Noble’s agar supplemented with 0.6% NaCl, 0.5 mM biotin, and 0.5 mM histidine). 2. 0.1 ml of bacterial culture 3. 0.1 ml of test agent in the appropriate solvent. ” [ Schechtman et al . 1978, p. 8]

“ Tubes were mixed quickly but gently and the contents poured over a base plate of Spizizen’s minimal medium.

.. Plates were rotated to distribute the overlay agar, allowed to harden andFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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80 incubated at 37 ° C for 48 hr at which time the number of mutant colonies were determined. ” [ Schechtman et al . 1978, p. 8]

“ Each agent was tested at five concentrations against all five strains of bacteria. ” [ Schechtman et al . 1978, p. 8]

“ Three plates were made for each control and each dilution of test agent. ” [ Schechtman et al

. 1978, p. 8] 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. ” [ Schechtman et al . 1978, p. 8]

“ The solvent was determined by the solubility of the test agent. Whe r ever possible, distilled water was the solvent of choice. DMSO, ethanol, acetone and ethyl acetat e may also be used in descending order of preference. The compound tested in this study was dissolved in DMSO. ” [ Schechtman et al . 1978, p. 8]

Non

- specific induction of liver enzymes: “ Aroclor 1254, a polychlorinated biphenyl, was used for the non

- specif

ic 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 CO 2 asphyxiation and the livers were removed and the S

- 9 fraction prepared. Animals had free access to food until 12 to 15 hr before sacrifice; water was available at all times. ” [ Schechtman et al . 1978, p. 9]

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 KC l and homogenized in a Potter Elvejhem tissue grinder at 120

- 150 rpm. The homogenate was centrifuged at 9 0 00 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 us e. ” [ Schechtman et al . 1978, p. 9]

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 MgCl 2 , 0.33 mM KCl, 0.5 mM glucose 6 phosphate, 0.4 m M NADP, and 100 mM NaH 2 PO 4 *H 2 O

, pH 7.4. Reagents for the S

- 9 mix were weighed and prepared fresh daily withFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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81 the exception of sodium phosphate which was made as a stock solution of 0.2 M. The entire S

- 9 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. ” [ Schechtman et al

. 1978, p. 9]

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

. ..” [ Schechtman et al . 1978, p. 9]

“ 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. ” [ Schechtman et al

. 1978, p. 9]

“ The activity of the S

- 9 preparation was confirmed by its ability to activate the control mutagens, 2

- AA and Aflatoxin B 1 which require metabolism to exert their effect, to reactive derivatives. ” [ Schechtman et al . 1978, p. 10]

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. ” [ Schechtman et al . 1978, p. 10]

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 B 1 TA1537: 1 μg 6

- Aminochrysene TA1538: 2 μg 2

- Aminofluorene TA98: 1 μg Aflatoxin B 1

“ Sterility controls on each d ilution 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. ” [ Schechtman et al . 1978, p. 10]

“ 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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82 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. ” [ Schechtman et al . 1978, p. 11]

“ 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. ” [ Schechtman et al . 1978, p. 12]

RESULTS

“... t he 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 T A98. ” [ Schechtman et al . 1978, p. 12]

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, T A1 538, TA100 and T A98. ” [ Schechtman et al . 1978, p. 12]

Genotoxicity result: Positive without metabolic activation

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. ” [ Schechtman et al . 1978, p. 13]

“ 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. ” [ Schechtman et al . 1978, p. 13]

“ 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. ” [ Schechtman et al . 1978, p. 13]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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83 STUDY RELIABILITY

[2] Reliable with restrictions. Important details were missing from the study including purity and composition of the test substance and GLP compliance.

REFERENCES

Schechtman, L., Parmar, A., and 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.

Text in quotes is taken directly from Schechtman et al . (1978).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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84 Study 12: Mutagenicity study

- In vitro gene mutation in mammalian cells ( Durward and Nolan, 2000)

Title: LZ1224.20: L5178Y TK +/

- Mouse Lymphoma Assay

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonyms: Diethyltoluenediamine (DETDA), DETDA 80

Remarks: 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 ; a mber slightly viscous liquid; Batch No. 2235

METHOD

Method/guideline followed: OECD TG 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 three

- fold 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 mutantsFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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85 DETDA would be reported equivocal if only one of the above criteria was met.

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 to 14 days and then scored for colony formation. The plating ef ficiency 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 veh icle 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 10 6 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 v essels 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 10 6 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 10 6 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 we re 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. “ Howeve r, in experiment 1, there were only three dose levels that were not too toxic, and in e xperiment 2, optimum toxicity (10 to 20% survival) was not achieved. Therefore, a third confirmatory experiment was performed to investigate the reproducibility of the response. ” [Albemarle Corporation 2012, IUCLID p. 4] 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 10 5 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.

Th e remaining cells were incubated and subcultured daily for the required expression period, by counting and dilution to 2 x 10 5 cells/mL. On the second day after the end of the treatment period, the cells were counted, diluted to 10 4 cells/mL, and plated f or mutant frequency (2000Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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86 cells/well) in selective media containing 4 μg/mL 5

- trifluorothymidine (5

- TFT). After 10 to 14 days of incubation, t he plates were scored using a magnifying mirror box. To assist the scoring of the TFT mutant colonies, MTT solut ion 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 usi ng 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 10 6 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 10 6 mutant frequency per survivor are not normally acceptable and will be repe ated. ” [ Albemarle Corporation 2012, IUCLID p. 5] 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 with metabolic activation

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 o f 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 absen ce 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 theFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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87 absence of metab olic 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%. 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 (linea r trend) increase in the mutant frequency at 10 6 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 witho ut 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 rel ative 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, a nd 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 relati ve 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 obse rved 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 concentra tion

- 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 cl astogenic 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 th ymidine kinase locus in the presence of metabolic activation and should be considered positive in the mouse lymphoma gene mutation assay.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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88 STUDY RELIABILITY

[2] Reliable with restrictions. The report was supplied as an IUCLID summary and important detail s were missing. The summary defines the medium R10 but does not define medium R0 or 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 mut ant 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 mammali an 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. [As summarized in Albemarle Corporation , 2012.]

Albemarle Corporation . 2012. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT), US EPA (letter dated March 1, 2012). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0660 as of September 12 , 2014

. Text in quotes is taken directly from Albemarle Corporation (2012).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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89 Study 1 3 : Mutagenicity studies in Salmonella typhimurium (Naismith and Matthews, 1979 a )

Title: Ames Salmonella /microsome plate test

This study is : Adequate Not Adequate

This study is not adequate because the test substance is not adequately described and the purity and compo sition are not provided. The substance is referred to only as “DETA”.

TEST SUBSTANCE

Identity: Test substance is referred to only as “DETA”

.

Remarks: Purity not specified; d ark 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
μ g/plate. Strain TA1538 showed complete inhibition at 5000 μ g/plate and abnormal background lawn growth at all other levels. Strain TA100 showed complete inhibition at 5000 μ g/plate and moder ate inhibition at 1000 and 200 μ g/plate. Dose levels for DETA in the plate assay were 1000, 333, 100, 33, and 10 μ g/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. ” [Naismith and Matth ews 1979 a , p. 1]

“ 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:Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

90 TA1535

- without activation

- N

- Methyl

- NPR

- Ni tro

- 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

- Nitro soguanidine ” [Naismith and Matthews 1979 a , p. 2 of SOP ]

“ 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. ” [Naismith and Matthews 1979 a , pp. 2

- 3 of SOP ]

RESULTS

“ The results for DETA were negative in strains T A1535, 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. ” [Naismith and Matthews 1979 a , p. 1]

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 8 μ g/plate

Results remarks: “ All negative and positive controls were within one standard deviation of historical data. ” [Naismith and Matthews 1979 a , p. 1]

CONCLUSIONS

The test substance was not mutagenic in S. typhimurium strains T A1535, TA1537, TA1538, TA98 or TA100 with or without metabolic activation.

STUDY RELIABILITY

[3] Not reliable. The test substance is not adequately described and the purity and composition are not provided. The substance is referred to only as “DETA”. Other missing details include the statistical methods used.

REFERENCES

Naismith, R., and Matthews, R. 1979 a . Summary Data from Ames Salmonella /Microsome Plate Test With and Without Metabolic Activation on DETA. Performing Laboratory: Pharmakon Laboratories, Scranton, PA. Study No. PH

- 340/301

- ET

- 003

- 79; PH

- 301

- ET

- 007

- 79. Sponsor: Ethyl Corpo ration.

Text in quotes is taken directly from Naismith and Matthews (1979 a ).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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91 Study 1 4 : 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 report, including purity and composition of the test substance, method/guideline foll owed, 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 recommendati ons, 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 effi cacy of the S9

- mix. (3) Results of positive and negative control assays were not provided.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Referred to as Diethyltoluenediamine (DETDA) and MA #T4018 in the study report

.

Remarks: Purity not specified; 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 10

, 00 0 μg/plate (preliminary toxicity assay); test concentrations for the mutagenicity assay were not specified

Statistical methods used: None specified

Test method/conditions re marks: The experimental materials, methods and procedures are based on those described by Ames, B, N., et al

. Methods for detecting carcinogens andFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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92 mutagens with the Salmonella /Mammalian

- Microsome Mutagenicity Test, Mutation Research 31 : 347

- 364, 1975.

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. I mmediately 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. ” [Lawlor 1985, p. 3]

Top Agar for Viable Count Determination: “ Minimal top agar as described above was supplemented with 35 m l /100 ml agar of a sterile solution which contained 1.4 mM L

- histidine and 0.12 mM D

- biotin. ” [Lawlor 1985, p. 3]

Minimal Bottom Agar: “ Bottom agar was Vogel

- Bonner minimal medi um E. ” [Lawlor 1985, p. 3]

Nutrient Broth: “ Nutrient broth used for growing overnight cultures of the tester strains was Vogel

- Bonner salt solution supplemented with 25 g per liter of Nutrient Broth No. 2 (Oxoid). ” [Lawlor 1985, p. 3]

Nutrient Bottom Agar: “ Nutrient bottom agar was Vogel

- Bonner minimal medium E supplemented with 2 5 g per liter of Nutrient Broth No. 2 (Oxoid). ” [Lawlor 1985, p. 3]

Tester Strain Diluent: “ Diluent for tester s train titering contained Vogel

- Bonner salt sol ution with 25 g per liter of Nutrient Broth No. 2 (Oxoid). ” [Lawlor 1985, p. 4]

Test Article Diluent: “ The solvent used for diluting the test article was dimethylsulfoxide (CAS #67

- 68

- 5 ) , Fisher Scientific Company, Certified A.C.S. ” [Lawlor 1985, p. 4]

T ester Strains: “ The tester strains used were the histidine auxotrophs TA98, TA100, TA1535, TA1537

, and TA1538 as described by Ames. ” [Lawlor 1985, p. 4]

“ 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 nu mber 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 nu mber of revertants per plate and/or produced a thinning or disappearance of the background bacterial lawn. ” [Lawlor 1985, pp. 5

- 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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93 “ When necessary, separate dose levels are plated for the portion of the assay with metabolic activation and the portion of t he assay without metabolic activation. ” [Lawlor 1985, p. 6]

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 le vel and activation as described in detail in Microbiological Associates, Inc. ’ s Microbial Mutagenesis Standard Operating Procedures. ” [Lawlor 1985, p. 6]

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 teste r strains (TA98, TA100, TA1535, TA 1 537 and TA1538) with metabolic activation and with out 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 aga r 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 minima l 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. ” [Lawlor 1985, pp. 6

- 7]

Positive Controls: “ All combinations of positive controls and tester strains plated along with the assay are listed below ”. [Lawlor 1985, p. 7] :

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 th e individual strain data forms. ” [Lawlor 1985, p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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94 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. ” [La wlor 1985, p. 7]

Preparation of S

- 9 Homogenates: “ Liver microsomal enzymes were prepared from male Sprague

- Dawley rats that had been injected with Aroclor 1254 at 500 mg/kg. The Aroclor was diluted in 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. ” [Lawlor 1985, pp. 7

- 8]

“ 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 min ced 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. ” [Lawlor 1985, p. 8]

Preparation of S

- 9 Mix: “ The S

- 9 mix was prepared immediately before its use in the mutagenicity assay. ” [Lawlor 1985, p. 8]

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. ” [Lawlor 1985, pp. 8

- 9]

“ 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 s train data forms. ..” [Lawlor 1985, p. 9]

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. ..” [Lawlor 1985, p. 9]

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. ” [Lawlor 1985, p. 10]

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. ” [Lawlor 1985, p. 10]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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95 RESULTS

G enotoxicity result: Positive

Cytotoxic concentration (if applicable): Not indicated

Results remarks: None specified

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. ” [Lawlor 1985, p. 2]

STUDY RELIABILITY

[3] Not reliable. Important details are missing from the study report, including purity and composition of the test substance, method/guideline followe d, 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 and negative 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.

Text in quotes is taken directly from Lawlor (1985).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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96 Study 1 5 : 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 tes t substance is identified as “diethyl toluene amine”, which is not the chemical specified in the test rule and is not considered an appropriate analog for the test rule chemical.

TEST SUBSTANCE

Identity: Diethyl toluene amine ( DETA ); also referred to as T1566 in the study report.

Remarks: Purity: 94%; 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 tri plicates were averaged and the standard deviation cal culated. 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 con sidered for the increase must have an average number of re vertant colonies which is three times that of the solv ent control. ” [ Schechtman et al

. 1980, p. 9 ]

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. ” [ Schechtman et al . 1980, p. 3]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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97 A. Mutagenesis Assay Without Metabolic Activation: “ The assay was performed by the method of Am es et al

... with the addition of several recomm endations

. .. 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 10 8 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 μ l) was added. The tubes were vortex

- mixed and the contents po ured 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 w ere 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. ” [ Schechtman et al . 1 980, pp. 6

- 7]

“ 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

com pensate 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 ) X 2 + (6.31 x 10

- 7 ) X 3 , where Y = corrected counts and X = machine counts, was found to transform the machine counts to values which accurately reflected manual coun ts

. 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). ” [ Schechtman et al . 1980, p. 7]

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 a septically 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 co ntent prior to use. ” [ Schechtman et al . 1980, p. 7]

“ 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 MgCl 2 , 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. ” [ Schechtman et al . 1980, p. 7]

“ The mutation assay was performed in the same way as that described in [previously]... , 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
p erformed 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 coun ted on a Biotran II automatic colony co unter

. All machine countsFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

- 98

- 1

98 between 12 and 1935 were corrected by the following formula: Y =
- 7.4 + 1.606X

- (7.1 x 10

- 4 ) X 2 + (6.31 x 10

- 7 ) X 3 , where Y = corrected counts and X = machine counts. No correction was required for machine counts less than 12 and all machine counts greater than 1935 were reported
as too numerous to count (TNTC)

. ” [ Schechtman et al . 1980, p. 8]

Controls: “ In the assay without exogenous metabolic activation, so dium azide (30 μg/plate) was used as the positive control for TA1535 and TA100, 9

- aminoacridine (100 μg/plate) for T A1537, and 2

- nitrofluorene (5 μg/plate) for TA1538 and TA98. For the assay with exogenous metabolic activation, 2

- aminoanthracene ( 2.5 μg/p late) 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. ” [ Schechtman et al . 1980, p. 8]

Criteria for Determination of a Valid Test: “ All sterility c ontrols, including the five concentrations of the test compound and S

- 9 mix, must be negative for bacterial growth .” [ Schechtman et al . 1980, p. 8]

“ 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. ” [ Schechtman et al . 1980, p. 8]

“ 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. ” [ Schechtman et al . 1980, p. 9]

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. ” [ Schechtman et al . 1980, p. 10]

“ The sterility controls for the S

- 9 mix, positive controls, and the tes t article dilutions were all negative for bacterial contamination. ” [ Schechtman et al . 1980, p. 10]

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. ” [ Schechtman et al . 1980, p. 13]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

99 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.

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 2 bacterial strains were tested and bec ause the test substance is identified as “diethyl toluene amine”, which is not the chemical specified in the test rule and is not considered an appropriate analog for the test rule chemical . In addition, important study details were missing from the study , including the full composition of the test substance, GLP compliance and test method/guidelines followed

.

REFERENCES

Schechtman, L., Curren, R., and Pa r mar, A. 1980. Activity of T1566 in the Salmonella / Microsomal Assay for Bacterial Mutagenicity Final Report. Performing Laboratory: Microbiological Associates. Bethesda, MD. Sponsor: Ethyl Corporation.

Text in quotes is taken directly from Schechtman et al . (1980).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

100 Study 1 6 : 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. In addition, i mpo rtant study details were missing from the study summary, including purity and composition of the test substance, statistical methods used

, and whether or not solvent and positive controls responded appropriately.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as darkened DETDA

- low color in the study report.

Remarks: Purity not specified; 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, 75, 37.5, and 10 mg/mL. ” [ Johnson and Petti grew 1987b , p. 4] “ 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. ” [ Johnson and Pettigrew 1987b , p. 4]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

101 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

- L ow C olor did not cause a dose related increase in mutant colonies in any oth er strain in the presence of metabolic activation. ” [ Johnson and Pettigrew 1987b , p. 4]

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. ” [ Johnson and Pettigrew 1987b , p. 4]

CONCLUSIONS

“ Darkened DETDA

- Low Color was not genetically active in the Salmonella / Microsomal Assay according to Protocol

- Ettox 027. ” [ Johnson and Pettigrew 1987b , p. 4]

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 4 bacterial strains were tested. In addition, i mportant study details were missing from the study summary, including purity and composition of the test substance

, statistical methods used

, and whether or not solvent and positive controls responded appropriately

.

REFERENCES

Johnson, D., and Pettigrew, A. 1987b. Genetic toxicology. Salmonella /Microsomal Assay w ith Darkened DETDA

- Low Color (Lot #6198

- 20

- 1). Ames 101

- #112. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Johnson and Pettigrew (1987b).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

102 Study 1 7 : 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 and the test

was co nducted only in the presence of metabolic activation

. In addition, important study details were missing from the study summary provided and included purity and composition of DETDA

- Low Color, detailed methods, statistical methods used

, cytotoxicity of test substance and whether or not solvent and positive controls responded appropriately

.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediame (DETDA)

Remarks: Two test substances were used: DETDA

- Low Color, Lot # 5869

- 75

- 1 (clear liquid, composition not available) and DETDA

- Obg, Lot # 5868

- 1; Composit i on of DETDA

- Obg, Lot # 5868

- 1 : 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 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 (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, 7

, 500, 3

, 750, 1

, 000, and 750 μg/plate. DETDA

- Low Colo r was soluble at all dose levels tested. Each dose was treated in triplicate. An untreated control, solvent control and positive control were treated concurrently. ” [ Johnson and Pettigrew 1987c , p. 4]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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103 DETDA

- Obg (Lot #5868

- 1) was tested in the same strai ns and at the same dose levels as DETDA

- Low Color.

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. ” [ Johnson and Pettigrew 1987c , p. 4]

Genotoxicity result: Positive with metabolic activation

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. ” [ Johnson and Pettigrew 1987c , p. 4]

CONCLUSIONS

“ DETDA

- Low Color was not genetically active in the Salmonella / Microsomal Assay according to Protocol

– Ettox 027. ” [ Johnson and Pettigrew 1987c , p. 4]

DETDA

- Obg induced gene mutations in the presence of metabolic activation.

STUDY RELIABILITY

[3] Not reliable. This study is considered to be unreliable because only 4 bacterial strains were tested and the test was conducted only in the presence of metabolic activation. In addition, important study details were missing from the study summary provided and included purity and composition of DETDA

- Low Color, detaile d methods, statistical methods used, cytotoxicity of test substance and whether or not solvent and positive controls responded appropriately

.

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.

Mulholland, A. 1986. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Johnson and Pettigrew (1987c).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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104 Study 1 8 : 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. In addition, impo rtant study details were missing from the study summary provided and included purity and composition of the test substance.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified ; 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 so lvent 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 solven t control is within the 95% confidence limits of the historical mean for control values... and the test chemical produces an increase greater than or equal to three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a two

- fold increase in the number of histidine

independent revertants.” [ Stankowski and Naismith 1987b , p. 4]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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105 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 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 as a concurrent positive control was a second sample of DETDA (Dr um #6131). This latter sample of DETDA was previously found to be mutagenic in the Ame s/ Salmonella Plate Incorporation Assay. ” [ Stankowski and Naismith 1987b , p. 5]

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 article , positive control or solvent in sterile glass tubes preheated to 45°C. The S9 mixture contained 8mM MgCl 2 , 33mM KC l , 4mM NA DP, 5mM glucose

- 6

- phosphate, 100mM NaH 2 PO 4 (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. W ithin an hour the plates were inverted and incubated in the dark at 37°C for 48

- 72 hours. ” [ Stankowski and Naismith 1987b , p. 4]

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 mi x 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. ” [ Stankowski and Naismith 1987b , p. 4]

Scoring: “ Following the 48

- to 72

- 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 conflu ent 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. ..” [ Stankowski and Naismith 1987b , p. 4]

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 cul tures

. 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. ” [ Stankowski and Naismith 1987b , p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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106 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. ” [ Stankowski and Naismith 1987b , p. 5]

CONCLUSIONS

T he “ 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. ” [ Stankowski and Naismith 1987b

, p. 6]

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 t he 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.

Text in quotes is taken directly from Stankowski and Naismith (1987b).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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107 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 c omposition 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

- die thyl

- ar

- methyl

- , CASRN 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, and 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 num ber 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≥n 1 /n) = (n!/(n 1 !(n

- n 1 )!)) (P n1 (1

- P) n

- n1 )Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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108 Where p is the probability, n is the number of induced mutants, n 1 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 induc ed mutation frequency is considered significant at the p ≤ 0.05 level. ” [ Schechtman et al . 1982, pp. 5

- 6]

Test method/conditions remarks: “ Exponentially grown BALB/3T3 clone A31

- 1 cells were seeded for each treatment condition at approximately 2 x 10 6 cells/75 cm 2 flask and were incubated at 36oC in a humidified atmosphere of 5% CO 2 in air for 20

- 24 hours. ” [ Schechtman et al . 1982, p. 4]

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

con trols 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 wer e washed once with HBSS, collected by trypsinization, and subcultured to assess cytotoxicity and to initiate the phenotypic expression period. ” [ Schechtman et al . 1982, pp. 4

- 5]

“ For evaluation of cytotoxicity, cells from each treatment condition were s ubcultured 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. ” [ Schechtman et al . 1982, p. 5]

“ For expression of the mu tant phenotype, cells from each treatment condition were subcultured in complete EMEM at a density of approximately 4 x 10 5 cells/150 cm 2 flask. After a 4

- 6 day expression period, selection for the mutant phenotype was performed. ” [ Schechtman et al . 1982, p. 5]

“ 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 10 5 cells/60 mm dish in complete EMEM containing 1 mM ouabain. After 1

- 4 weeks, the mutan t 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 c ounted for cloning efficiency. ” [ Schechtman et al . 1982, p. 5]

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 utiliz ed 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 o btained with one mutant colony. ” [ Schechtman et al . 1982, p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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109 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 m ust be equal to or greater than 40% for at least one dose. The number of spontaneous ouabain

- resistant 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. ” [ Schechtman et al . 1982, p. 6]

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 ouabain

resistant mutant colonies w ere 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 st atistically increased (p>0.05) relative to the solvent control. MNNG induced 247 ouabain

resistant mutants for a mutation frequency of 217 X 10

- 6 which was statistically significant (p<0.01) to the solvent control. ” [ Schechtman et al . 1982, p. 7]

“The n egative control and positive control fulfilled the requirements for determination of a valid test.” [ Schechtman et al . 1982, p. 10]

CONCLUSIONS

“ 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. ” [ Schechtman et al . 1982, p. 10]

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 A3 1

- 1 mouse cells) are not named in OPPTSFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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110 870.5300 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., Putman, D., an d 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 Corporati on, Baton Rouge, LA.

Text in quotes is taken directly from Schechtman et al . (1982).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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111 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, whi chever 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 ; Referred to as T1364A in the study report.

Remarks: Purity not specified; 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 10 4 , 1:1.5 x 10 4

, and 1:2 x 10 4 ( Concentrations were presented in the study report as dilutions of the test substance in reaction buffer, which were prepared by diluting the stock directly into reaction buffer. Actual concentrations are unk nown because the test substance purity and composition were not provided.)

Statistical methods used: Not indicatedFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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112 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 a m o dication of published procedures. ” [ Schechtman et al . 1979, p. 6]

“ Exponentially growing BALB/3T3 Cl.A31 cells were cultured in 75 cm 2 cell culture flasks and collected by tryp sinization 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. ” [ Schechtman et al . 1979, p. 6] 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 g rowth 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 3 7oC, 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

... . ” [ Schechtman et al . 1979, p. 7]

“ The remaining cells for each test condition were cultured further (in 150 cm 2 cell culture flasks) at 37oC for a 4

- 6 day 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. ” [ Schechtman et al . 1979, p. 7]

“ For determination of mutation induction, cells from each test condition collected post

expression period were also seeded at 2 X 10 5 cells per 60 mm dish, approximately 15 dishes per condition, and incubated at 37oC for 4 hours. One ml of 5 mM ouabain was then added to each dish, result ing 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 th e development of ouabain

- resistant colonies

... , according to the protocol of Schechtman and Kouri

... . ” [ Schechtman et al . 1979, p. 7]

“ The results of the mutation assays are expressed in terms of the number of ouabain

- resistant mutants per number of CAR, or a s the total number of ouabain

- resistant mutant colonies per condition. ” [ Schechtman et al . 1979, p. 7]

“ A preliminary cytotoxicity study was conducted to determine the dose range to be employed in the mutagenesis assay.

.. Whe n ever possible, the approximate LD 50 concentration of the test chemical plus one dose above and one dose below the LD 50 were those used in the mutagenesis
assay. ” [ Schechtman et al . 1979, p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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113 The following text is taken directly from Appendices A

- C of the study report:

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 10 6 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 t ubes containing each reaction mixture are incubated for 2 hr at 37C in a humidified atmosphere of 5% CO 2 + 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 resusp ended 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 numb er 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 solvent

treated controls).

Appendix B Standard Protocol for the Mutagenesis of BALB/3T3 Cl. A3 1 Mouse Cells at the Ouabain

Resistance Locus (Oua R ) in the Presence of Exogenous Metabolic Activation: Suspension AssayFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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114 1. Two

- 3 x10 6 logarithmically growing BALB/3T3 Cl. A31 cells (1) from sub

- confluent stock cultures are collected onto 50 cc capacity c onical 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 (ap proximately 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
at mosphere of 5% CO 2 + 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 superna tants 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 (deri ved 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

- 1 0 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 se eded into 150 cm 2 tissue culture flasks (~5 x 10 5 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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115 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 the selective agent (which discriminates mutant cells). These dishes are handled as described in step #6 above.

10. For mutagenesis determinations, 2 x 10 5 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 ster oid 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 oua bain 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 o f 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% CO 2 + air.

2. Twenty

- four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle (negat ive 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 desira ble, 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 wi th 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 relativ e plating efficiency (RPE) (i.e. number of surviving colonies per number of cells seeded) and relative colony

- forming efficiency (RCE) (i.e. numberFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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116 of colonies in chemically

- treated dishes relative to the number of colonies in solvent

- treated controls).

7 . The approximate LD 50 is determined from the relative survival data. The LD 50 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 10 4 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 10 4 , 1:1.5 x 10 4 and 1:2 x 10 4 , respectively.

Results remarks: Under the conditions of the preliminary cytotoxicity study, “ 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. ” [ Schechtman et al . 1979, p. 8]

In an additional preliminary cytotoxici ty assay utilizing revised dilutions, “ T1364A was observed to be 100% cytotoxic at the 1:1 x 10 3 , 1:2 x 10 3 and 1:3 x 10 3 dilutions. Although cell survival and colony formation was observed at the 1:5 x 10 3 dilution, the relative survival of the target ce lls at this concentration was judged to be too low to be used in the mutation assay. ” [ Schechtman et al . 1979, p. 8]

“ These results necessitated an additional cytotoxicity assay which would serve to estimate the approximate LD 50 of the test compound in th e 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 surviva l after treatment could be established. Under these conditions, compound T1364A was again observed to be extremely cytotoxic, even at a 1:1 x 10 4 dilution (relative survival ≤ 10%). A dramatic decrease in the level of toxicity was observed at dilutions ≥ 1:1.5 x 10 4 . The observed cellular response made it difficult to establish an LD 50 for test agent T1364a. Therefore, the mutagenesis assay (and its accompanying cytotoxicity assay) was performed using these same doses in order to bracket the LD 50 range. ” [ Schechtman et al . 1979, p. 8]

Under the conditions of the quantitative cytotoxicity study which accompanied the mutagenesis
assay, “ an approximate LD 60 was observed at the 1:1 x 10 4 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. ” [ Schechtman et al . 1979, p. 8]

Under the conditions of the mutagenesis assay, “ no ouabain

- resi stant 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 colo nies per 0.58 x 10 6 cells at risk. ” [ Schechtman et al . 1979, p. 8]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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117 The negative controls, positive controls, and sterility controls all fulfilled requirements for determination of a valid test.

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 metabo lic 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 0.01 M, 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, t he test substance is identified as diethyltolueneamine, which differs from diethyltoluenediamine (the subject of required testing); diethyltolueneamine is not an acceptable analog for diethyltoluenedia mine.

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. Sponso r: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Schechtman et al . (1979).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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118 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 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. Ad ditionally, the study did not indicate the test guideline(s) followed, GLP compliance, or test substance purity and composition.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediame (DETDA) ; Referred to as “DETA” and identified as “diethyltoluenediamine” in the study report.

Remarks: Purity not specified; L ot 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 cm 2 Falcon flasks and collected by trypsinization. Cells were seeded in triplicate dishes per condition at densities of 250 an d/or 500 cells per 60 mm dish for determination of the relative cytotoxic effects of each treatment and 1 x 10 4 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. ” [ Schechtman et al . 1978, p. 6]

“ 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 inFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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119 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 HBSS [Hanks’ Balance Salt Solution] 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). ” [ Schechtman et al . 1978, p. 6]

“ Six days after the initiation of the experiment, the dishes which were initially seeded with 1 x 10 4 cel ls were treated with medium containing 1 mM ouabain for selection of resistant mutant colonies developing over 25

- 30 days. ” [ Schechtman et al . 1978, p. 6]

“ Cells were fixed with 100% methanol and stained with 10% aqueous Giemsa after 8

- 10 days for cytotox icity and plating efficiency (cells at risk) determinations, according to the protocol of Schechtman and Kouri

... , and at 25

- 30 days for determination of the number of mutant colonies in each dish, according to the protocol of Schechtman and Kouri

... . Plati ng 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

- resistan t mutants per number of cells at risk to ouabain. ” [ Schechtman et al . 1978, p. 7]

“ A preliminary cytotoxicity study was conducted to determine the dose range to be employed in the mutagenesis assay.

... Wherever possible, the approximate LD 50 concentration of the test chemical plus 1 dose above and 1 dose below the LD 50 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 contro l. ” [ Schechtman et al . 1978, p. 7]

The following text is taken directly from Appendi ces A and B of the study report:

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% CO 2 + 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 t o 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 me dium.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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120 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 (R PE) (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 Ouabain

Resistance Locus (Oua R ) 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 10 4 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% CO 2 + air.

2. Twenty

- four hours la ter, 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 use d 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 f or 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 t he 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 represent ative 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).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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121 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 numb er 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 DETA 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 DETA precipitated heavily at all of the
concentr ations tested. ” [ Schechtman et al . 1978, p. 8]

In an additional preliminary study using revised dilutions, “ DETA 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. ” [ Schechtman et al . 1978, p. 8]

In the cytotoxicity study which accompanied the mutagenesis assay, a dose response was observed. “ Although DETA 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 LD 40 was found to be at the 1:30000 dilution of compound DETA. ” [ Schechtman et al . 1978, p. 8]

In the mutagenesis assay, “ DETA was effective in inducing oua bin

- 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 o bserved 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) induce d 20 ouabain

- resistant colonies per 6.5 x 10 6 cells at risk

. ” [ Schechtman et al . 1978, p. 8]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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122 CONCLUSIONS

“ Based on the results and discussion the following conclusions were made: (1) DETA was found to be extremely cytotoxic to the BALB/3T3 Cl. A31 c e lls. (2) DETA precipitated in the 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 bioassay, the test chemical DETA was demonstrated to be positive in pr oducing 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). ” [ Schechtman et al . 1978, p. 13]

STUDY RELIABILITY

[3] Not reliable. 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 metabo lic activation. Additionally, the study did not indicate the test guideline(s) followed, GLP compliance, or test substance purity and composition.

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.

Text in quotes is taken directly from Schechtman et al . (1978).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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123 D

- 7 . Genetic Toxicity

– Chromosomal Aberrations

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

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonyms: Diethyltoluenediamine (DETDA), DETDA 80

Remarks: 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; Clear liquid; Batch No. 2235

METHOD

Method/guideline followed: OECD TG 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.

“ 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 activat ion. ” [Albemarle Corporation 2012, IUCLID p. 4] TheFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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124 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 selec ted 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

- hou r 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.

Expe riment 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 metaph ase analysis were: 1400 and 1800 μg/mL.

Cell Harvest: “ Mitosis was arrested by addition of demecolcine

... two hours before the required harvest time. ” [Albemarle Corporation 2012, IUCLID p. 4] 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 ran domly. “ 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. ” [Albemarle Corporation 2012, IUCLID p. 5] 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 freque ncy, a concentration

- response 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 ce lls were compared, where necessary, with the concurrent vehicle control value using Fisher’s exact and C hi

- squared tests. ” [Albemarle Corporation 2012, IUCLID p. 5]

Vehicle Used?: Yes

Vehicle Name: DMSO

Positive Control Substances: Without metabolic activation: ethylmethanesulphonate (Experiment 1) and mitomycin C (Experiment 2). With metabolic activation: cyclophosphamide.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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125 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 th e 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

- t ype 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.

Expe riment 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 Experimen t 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 excess ive 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 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 differenceFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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126 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 polyploid y 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 indu ced 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 concentration

related. This response was not reprod uced 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% S 9 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 activa tion, 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 n umber of cells with chromosomal aberrations and types of chromosomal aberrations for control and treated cultures.

REFERENCE S

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. [A s summarized in Albemarle Corporation , 2012.]

Albemarle Corporation . 2012. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT), US EPA (letter dated March 1, 2012). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0660 as of September 12 , 2014

. Text in quotes is ta ken directly from Albemarle Corporation (2012).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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127 Study 2: Micronucleus study in mice (Durward and Nolan, 2000)

Title: LZ1224.20: Micronucleus Test in the Mouse

Th is study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonyms: Diethyltoluenediamine (DETDA), DETDA 80

Remarks: 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 ; Amber slightly viscous liquid; Batch No. 2235

METHOD

Method /guideline followed: OECD TG 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, and 500 mg/kg

- bw test mat erial; 50 mg/kg

bw 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

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128 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 treatme nt, 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 co ntrols 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 evaluatio n 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 N CE 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: “[ P ] remature 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. ” [Albemarle Corporation 2012, IUCLID p. 5]

DETDA “ showed no marked difference in its toxicity to male or female mice. Therefore, males were used for the definitive study. Adequa te 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. ” [Albemarle Corporation 2012, IUCLID p. 5]

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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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129 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 subs tance.

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 S

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. [A s summarized in Albemarle Corporation , 2012.]

Albemarle Corporation . 2012. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT), US EPA (letter dated March 1, 2012). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0660 as of September 12 , 20 14

. Text in quotes is taken directly from Albemarle Corporation (2012).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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130 Study 3 : Micronucleus study in mice (Naismith and Matthews, 1979 b )

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 ho urs 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 wherea s CFR 799.9539 stipulates the scoring of at least 2000/animal.

TEST SUBSTANCE

Identity: Benzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediame (DETDA) ; Referred to as “DETA” and identified as “diethyltoluenediamine” in the study report

Remarks: Purity not specified ; 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

- bwFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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131 Statistical methods used: Student’s “t” test

Test method/conditions remarks: “ In a preliminary dose

- range study, DETA 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. ” [ Naismith and Mathews 1979 b

, p. 1] 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, DETA 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% methylc ellulose (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 Microscop e. Results are analyzed according to the Student’s “t”
test. ” [ Naismith and Mathews 1979 b

, p. 5 of SOP ]

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. ” [ Naismith and Mathews 1979 b

, p. 1]

“ The 250 mg/kg dose group exhibited ataxia, complete bod y 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. ” [ Naismith and Mathews 1979 b

, p. 1]

“ 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. ” [ Naismith and Mathews 19 79 b

, p. 1]

“ The 100 mg/kg dose group exhibited body drop, loss of body tone, ptosis, depression, abnormal gait, and decreased spontaneous activity. No dea ths occurred. ” [ Naismith and Mathews 1979 b

, p. 2]

“ The 50 mg/kg dose group exhibited decreased spontaneous activity

, slight loss of body tone and depression. No deaths occurred. ” [ Naismith and Mathews 1979 b

, p. 2]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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132 Micronucleus Test: DETA 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.

Gen otoxicity result: Negative

Cytotoxic dose: Not indicated

Results remarks: The positive control performed as expected. “ As per the sponsor’s request, slides from the Micronucleus Test on DETA were rescored in 1984 according to present criteria. The slides were first counted in 1979, prior to changes in the criteria for scoring micronuclei. ” [ Naismith and Mathews 1979 b

, addendum p. 1]

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, t his 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 follow ing 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 S

Naismith, R. and Matthews, R. 1979 b . Micronucleus Test (MN T). Study No. PH

- 309

- ET

- 004

- 79. Performing Laboratory: Pharmakon Laboratories, Scranton, PA. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Naismith and Mathews (1979 b ).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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133 D

- 8. Reproductive Toxicity

Summary of endpoint: A dequate

Although no reproductive toxicity studies were available, the reproductive endpoint has been acceptably addressed on the basis that an adequate developmental toxicity study is available and

no treatment

- related histopathological effects in reproductive organs were observed at sublethal dietary concentrations of CASRN 68479

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- 1 in adequate 90

- and 28

- day repeated dose toxicity studies, as well as a two

- year cancer study

. 1

1 Histopathological effects in reproductive organs were only observed in a 90

- day dietary study at a dose that resulted in 68% mortality.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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134 D

- 9. Developmental Toxicity

Summary of endpoint: Adequate

Study 1: Prenatal developmental toxicity study in rats ( Theuns

- van Vliet and Otto, 2014)

Title: An oral prenatal developmental toxicity study with diethylmethylbenzene diamine (DETDA) in rats

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: B enzenediamine, ar, ar

- diethyl

- ar

- methyl

- , CASRN 68479

- 98

- 1; referred to as diethylmethylbenzene diamine

, DETDA

, and Ethacure 100 in the study report.

Remarks: 97.8% pure; liquid, clear yellow; Batch No. 4500051816

- 011110; Composition: 2,4

- DETDA (77.8%), 2,6

- DETDA (20.0%), 2,4,6

- TEMPDA (0.3%), METDA (1%), TAPDA (0.68%).

METHOD

Method/guideline followed: OECD TG 414 (2001) “Prenatal Developmental Toxicity Study”; Method B.31 of Commission Regulation (EC) No. 440/2008 “Prenatal Developmental Toxicity Study”

Test type: Prenatal Developmental Toxicity Study

- Rats

GLP compliant?: Yes

Year study performed: 2013

Test species: Wistar rats (RccHan: WIST strain) from Harlan, Horst, the Netherlands; Body weight at initiation: 207.5

- 268.1 grams; Age at initiation of mating: ~12 weeks

Number of animals/sex/dose: 24 mated females/dose

Route of administration: Oral (diet)

Exposure duration: Continuous during gestation days 0

- 20

Doses/concentrations tested: Nominal dietary concentrations: 0 (control), 50, 150, and 500
ppm 1 ; estimated doses: 0, 2.63, 7.83, and 20.45 mg/kg

- bw/day ( based on initial measured

1 Dietary concentrations were selected based on the results of two dose

- range finding studies with exposure to the test item in pregnant rats (TNO Triskelion Report No. V20192DRF, October 2012 and TNO Triskelion Report No. V20193DRF2, June 2013). The dose

- range finding studies were not available for review.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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135 concentrations of test item in the diet, adjusted for test item loss during 3

- 4 day periods in the animal room ) 1

Statistical methods used: Two

- sided tests were generally performed and considered statistically significant if p < 0.05. For each endpoint, continuous or dichotomous decision trees 2 were consulted for data analysis. Statistical tests reported in the data tables included analysis of variance (ANOVA), Kruskal

- Wallis test, Dunnett’s test, Dunn’s test , Chi

- squared test, and Fisher’s exact probability test. The litter was used as the experimental unit for statistical analysis of fetal data.

Test method/conditions remarks: Test Item Preparation: The test substance was mixed into the basal diet with a blender, and then divided into individually wrapped amounts that were stored in a freezer (<

- 18 ° C). Portions of test diets were removed from the freezer twice per week (every 3

- 4 days) for administration to the animals. The prepared test diets were pr ovided to the animals in stainless steel cans, covered with a perforated stainless steel plate to prevent spillage.

Analyses of Test Diet Mixtures: The homogeneity, concentration, and stability of the test substance in the prepared test diets were determi ned in samples collected for analysis. Five samples of each freshly mixed test diet were collected directly from the mixer to determine the homogeneity and to confirm the test substance concentrations. Samples used to determine the stability of the test item in the prepared test diets were collected after 5 days of storage in an open container in the animal room, and after 31 days of storage in a closed container in a freezer (<

- 18 ° C). Analyses of test diets were performed by high performance liquid chr omatography

- mass spectrometry (HPLC

- MS) using a validated analytical method (Annex 7 of the study report describes the validation procedure). The measured concentration of test substance in the diet was considered to confirm the nominal concentration if t he difference between the mean measured concentration and the nominal concentration was < 10%.

Mating and Animal Allocation: Rats were housed in groups of 4 per sex during a 10

- day acclimation period. For mating, 112 females and 56 males were caged at a ratio of two females to one male. Vaginal examinations were performed each morning. The day on which sperm cells were observed in vaginal smears was considered gestation day (GD) 0. Females were caged individually after evidence of copulation and rando mly assigned to one of the four dose groups (24 females/dose group).

Exposure Conditions: Groups of mated female rats (24/group) were administered the test item continuously in the diet at nominal concentrations of 0 (control diet), 50, 150, or 500 pp m during gestation days 0 to 20.

1 During the study, feed available to the ani mals was removed e very 3

- 4 days and completely replaced by feed from the freezer. Since analytical measurements of the diets showed loss of the test item from feed over time in the animal room, the dose calculations were adjusted for test item loss during the 3

- 4 day feeding intervals.

2 Separate decision trees for dichotomous data and continuous data were provided in Annex 8 of the study report (“TNO Triskelion Study Plan”).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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136 Food and drinking water were available ad libitum throughout the study. The basal diet was a cereal

- based diet (RM3) from a commercial supplier.

Mortality and Clinical Observations: Animals were observed at least onc e daily during the dosing period for mortality and clinical signs of toxicity. Observations were performed cage

- side and animals were handled, if necessary, for determination of toxicity.

Body Weight and Food/Water Consumption: Individual body weights we re recorded on GDs 0, 3, 7, 10, 14, 17, and 20. The final body weight was recorded on GD 21.

Food consumption was determined for each female for the following time intervals: GDs 0

- 3, 3

- 7, 7

- 10, 10

- 14, 14

- 17, and 17

- 20. No water consumption data were pr ovided and the study report did not indicate whether water consumption was monitored.

Ophthalmic Examinations: The eyes of all females were examined with an ophthalmoscope (following induction of mydriasis by an atropine sulfate solution) before the mating period and on GD 20.

Hematology and Clinical Chemistry Analyses: Blood samples for hematology and clinical chemistry evaluations were collected from 5 animals per group at scheduled sacrifice (GD 21). All animals were fasted on the day prior to sacrifice. Hematological parameters that were evaluated included hemoglobi n, packed cell volume, red blood cell count, reticulocytes, total and differential white blood cells (lymphocytes, monocytes, neutrophils, eosinophils, basophils, and monocytes), thrombocyte count, prothrombin time, mean corpuscular volume, mean corpuscula r hemoglobin, and mean corpuscular hemoglobin concentration. Clinical chemistry parameters evaluated included glucose (plasma), alkaline phosphatase activity, alanine

- aminotransferase/glutamic

- pyruvic transaminase activity, aspartate aminotransferase/glut amic

- oxalacetic transaminase activity, gamma

- glutamyl transferase activity, total protein, albumin, ratio of albumin to globulin, urea, creatinine, bilirubin (total), cholesterol (total), triglycerides, phospholipids, inorganic phosphate, calcium, sodium, potassium, and chloride.

Gross Necropsy and Histopathology: All animals were sacrificed on GD 21 and examined for gross abnormalities at scheduled sacrifice. The pancreas of each dam was collected and preserved in a neutral, aqueous phosphate

- buffered 4 % solution of formaldehyde. For histological examinations, each preserved pancreas was embedded in paraffin wax, sectioned, and stained, and then examined by microscopy. No other tissues were examined microscopically. The uterus (containing placentas

an d fetuses or empty) and ovaries were weighed.

Cesarean Sections and Fetal Examinations: The uteri (including the fetuses), ovaries, and placentas of all females sacrificed on GD 21 were examined and the following parameters were recorded: number of corp ora lutea, number ofFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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137 implantation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, and number of grossly visible malformed fetuses and fetuses with external abnormalities. Live fetuses and their correspondi ng placentas were weighed. Placentas were also examined for gross abnormalities.

Fetuses were sacrificed after weighing. Half of the fetuses per litter were fixed in Bouin’s fixative and examined for soft tissue (i.e., visceral) anomalies. The remaini ng fetuses were examined for skeletal anomalies.

Reproductive Performance: For each dose group, the following indices were calculated and recorded: female fertility index,
pre

- implantation loss, post

- implantation loss, gestation index, and sex ratio.

RES ULTS

Endpoint value(s): The following values are based on initial measured concentrations of test item in the diet, adjusted for test item loss during 3

- 4 day periods in the animal room:

LOAEL (maternal toxicity) = 20.45 mg/kg

- bw/day, based on reduced mean maternal body weight and body weight gain, reduced mean food consumption, decreases in mean carcass weight and mean carcass weight change, and histological changes in the pancreas, including induction of pancreatic acinar cell apoptosis and mononuclear cell inflammation ; NOAEL (maternal toxicity) = 7.83 mg/kg

- bw/day.

LOAEL (developmental toxicity) = 20.45 mg/kg

- bw/day, based on reduced mean number of live fetuses per litter, reduced mean number of implantation sit es per dam, increased mean number of
pre

- implantation losses per dam, increased mean percentage of male fetuses per litter, decreased male and female fetal body weights, decreased placenta weight for female fetuses, and skeletal variations in fetuses, incl uding an increased incidence of unossified or incompletely ossified hindlimb metatarsals and incomplete ossification of vertebral sacral arches ; NOAEL (developmental toxicity) = 7.83 mg/kg

- bw/day.

Results remarks: Analyses of Test Item in Prepared Test D iets: The test item was found to be distributed homogenously in the test diets at all dose levels. In samples of test diet collected immediately after mixing, measured concentrations of test item (corrected for % recovery) 1 were 16%, 10%, and 15% lower t han the nominal concentrations of 50, 150, and 500 ppm, respectively. After 5 days of storage in an open container in the animal room, measured concentrations in the test diets were 33%, 41%, and 32% lower than the initial measured concentrations for the low, middle, and high dietary concentrations, respectively; therefore, the test substance was not considered to be stable in the test diet under the conditions of the animal room. Concentrations of the test item in frozen test diet samples were found to b e stable.

1 Measured concentrations were corrected for % recovery using recovery values obt ained during the validation test.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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138 Since the initial concentrations of the test substance in the diets were lower than intended and the test item concentrations in the diets were not stable under the conditions in the animal room, the study authors adjusted their dose calculation s to account for these factors. Doses were calculated using measured feed consumption values and initial measured concentrations of test substance in feed that were adjusted for estimated loss of the test substance during the feed consumption intervals (3

- 4 days). The study authors referred to each calculated dose as the “interpolated actual mean test substance intake”.

Mortality and Clinical Observations: No mortalities occurred during the study. On gestation days 20 and 21, piloerection was observed a t the low

- , mid

- , and high

- dose levels in 1, 3, and 8 animals, respectively. Soiled fur was observed at the mid

- and high

- dose levels in 1 and 2 animals, respectively. Piloerection and soiled fur were not observed in control animals.

Body Weight and Food/Water Consumption: Mean body weights were significantly (p < 0.01) lower in the high

- dose group throughout the study compared to controls. The body weight disparities in high

- dose animals relative to controls increased with the duration of dosing; mean body weights of high

- dose animals were approximately 6.4%, 7.9%, 8.7%, 11.7%, 14.3%, and 16.6% lower than controls on GDs 3, 7, 10, 14, 17, and 20, respectively. A mean body weight loss of approximately 8 g was observed in high

- dose animals during G Ds 0

- 3 (compared to a gain of ~10 g in controls). High

- dose animals gained weight thereafter, but continued to display significant (p < 0.05) reductions in body weight gain compared to controls (reduction: ~30

- 64%). A significant (p < 0.05) decrease in

m ean food consumption per animal was observed in the high

- dose group (reduction: ~27

- 43%) relative to the control group for all time periods analyzed (i.e., GDs 0

- 3, 3

- 7, 7

- 10, 10

- 14, 14

- 17, and 17

- 20). At the middle dose, a slight but significant (p < 0.0 5) reduction in food consumption was noted during GDs 7

- 10 compared to controls, but this finding was considered incidental since food consumption in this group was comparable to the control group for the other time periods.

Ophthalmic Examinations: No treatment

- related effects were observed during ophthalmic examinations . At the pre

treatment observation, a defective eyelid and corneal damage were observed in one animal at the high dose, which later resulted in a degenerated eye (observed on GD 20). Degeneration of the eye was not seen in any other animals and was therefore considered unrelated to treatment. At the end of gestation, a bilateral cataract was noted in one rat at the high dose but was considered to be an incidental finding. Additional observations seen on GD 20 at a relatively low incidence in treated animals, but not in controls, included focal lens opacity (0, 1, 1, and 3 animals in the control, low

- , mid

- , and high

- dose groups, respectively) and persistent pupillary membrane (0, 0, 1 , and 1 animals in the control, low

- , mid

- , and high

- dose groups, respectively), as well as clear circular shape in lens (bilateral; one high

- dose animal).

Hematology and Clinical Chemistry Analyses: There were no statistically significant or dose

- related differences in hematological and clinical chemistry parameters among groups.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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139 Gross Necropsy and Histopathology: No treatment

- related macroscopic findings were evident at necropsy. Mean carcass we ight (terminal body weight

– weight of full uterus) was reduced by ~10% in high

- dose animals relative to controls (p < 0.01). Also, mean carcass weight change (carcass weight at sacrifice

– body weight at GD 0) was significantly (p < 0.01) decreased in hi gh

- dose animals relative to controls; mean carcass weight increased by 7.07 g in controls but decreased by 18.54 g in high

dose animals. Significant (p < 0.05) decreases in absolute ovary weight (~12%), absolute uterus weight with fetuses (~37%), and abso lute empty uterus weight (~30%) were also observed in high

- dose animals compared to controls. (Relative organ weights were not reported.) The toxicological relevance of the organ weight changes is unknown in the absence of microscopic examination of thes e organs. The study authors attributed the changes in uterus weight to the decreased mean number of fetuses per dam at the high dose.

Statistically significant increases in the incidence of pancreatic histological findings were observed in the high

- dose group. Apoptosis of the pancreatic acinar cells was observed in 2/24 mid

- dose animals (not significantly different from controls) and 13/24 high

- dose animals (p < 0.01). The severity of apoptosis increased with dose, with minimal apoptosis observed in
t he pancreatic acinar cells of both affected mid

- dose animals, and minimal (4/24), mild (6/24), or moderate (3/24) apoptosis observed in the affected high

- dose animals. Mononuclear cell inflammation in the pancreas was observed in 1/24 1 mid

- dose animals (n ot significantly different from controls) and 15/24 high

- dose animals (p < 0.01). The severity of mononuclear cell inflammation was graded as minimal in the affected mid

- dose animal and as minimal (6/24) or mild (9/24) in the affected high

- dose animals. No additional histopathological effects were observed that were considered to be related to treatment.

Pregnancy Observations: Of the 24 females per group showing positive evidence of mating, 20, 19, 20, and 22 females in the control, low

- , mid

- and high

- dose groups, respectively, showed evidence of pregnancy. One of the high

- dose females that became pregnant did not possess live or dead fetuses at necropsy, but had one implantation site and a late resorption.

Examination of Uterine Contents: No dead fe tuses were found upon Cesarean section in any dose group. The total numbers of live offspring in the control, low

- , mid

- , and high

- dose groups were: 245, 230, 219, and 192, respectively. The mean number of live fetuses per litter (i.e., litter size) was significantly reduced (~29%; p < 0.05) at the high dose compared to the control group (mean litter sizes were 12.2, 12.1, 10.9, and 8.7 at 0, 2.63, 7.83, and 20.45 mg/kg

- bw/day , respectively).

The mean number of implantation sites per dam was 12.9, 12.7, 11.4, and 9.5 at 0, 2.63, 7.83, and 20.45 mg/kg

- bw/day , respectively, and was significantly reduced at the high dose compared to controls (reduction: ~26%; p < 0.05). The mean number of pre

- implantation losses per dam was 0.7, 0.9, 1.8, and 3.8, at 0, 2.6 3, 7.83, and 20.45 mg/kg

- bw/day, respectively, and was sig nificantly increased at the high dose compared to controls (increase: ~5

- fold; p < 0.01). The effects on numbers of live fetuses, implantation sites, and pre

- implantation losses were considered to be related to treatment.

1 A total of 3/24 mid

- dose animals exhibited histological effects in the pancreas

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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140 Fetal Examinations: Significant decreases of approximately 10 to 12% were observed for overall mean fetal body weight (p < 0.05), mean male fetal body weight (p < 0.05), and mean female fetal body weight (p < 0.01) in the high

- d ose group compared to the control group. Placenta weight of female fetuses was reduced by approximately 8.7% (p < 0.05) in the high

- dose group relative to the control group. Fetal placenta weights for both sexes combined and for males only were similar f or test substance

- exposed groups and controls.

The fetal sex ratio of the high

- dose group was statistically significantly different from that of the control group. The mean percentage of male fetuses per litter was 41.6%, 46.4%, 49.1%, and 56.8% at 0, 2.63, 7.83, and 20.45 mg/kg

- bw/day , respectively, and was significantly increased at the high dose compared to controls (increase: ~37%; p < 0.01). The study authors did not consider this effect to be related to treatment on the basis of an unusually low percentage of males in the control group versus historical control data (historical control data range: 41.6

- 52.7%). However, the control mean for this prenatal developmental toxicity study is within the historical control data range provided by the study authors; therefore, the possibility of a
treatment

- related effect on sex ratio cannot be dismissed.

Some fetuses in the mid

- dose (3 fetuses from 3 different litters) and high

- dose (15 fetuses from 2 different litters) groups were noticeably small in size. Additional external findings included two fetuses being pale in color (mid

- dose group only), and a low incidence of subcutaneous hemorrhage in all groups, including controls.

Significant (p < 0.01) decreases in both the number of litters with visce ral anomalies (reduction of 45%) and the percentage of fetuses per litter with visceral anomalies (reduction of ~54%) were observed in the high

- dose group compared to the control group. The study authors did not discuss possible reasons for a reduction in visceral anomalies at the high dose. Visceral anomalies included misshapen lens, hole in lens, hernia ventralis, dilated ventricles of the brain, discolored stomach contents, blood

- filled pericardium, folded retina, variations in the ureters, distended u rinary bladder, and dilation of the renal pelvis. None of these anomalies showed a positive dose

- response pattern (i.e., increase in incidence as dose increased) and, therefore, none were considered to be toxicologically significant.

Certain skeletal variations were observed at an increased incidence in the high

- dose group. A significant (p < 0.01) increase in the incidence of fetuses per litter with 1 to 5 unossified or incompletely ossified hindlimb metatarsals was observed in the high

- dose group c ompared to the control group (99.0% vs. 86.0%, respectively). The incidence of fetuses per litter with 1 or 2 unossified hindlimb metatarsals was significantly increased in the high

- dose group compared to the control group (68.7% vs. 35.5%, respectively; p < 0.05). An increased incidence of litters with incompletely ossified vertebral sacral arches was observed in the high

- dose group compared to the control group (61.9% vs. 20%, respectively; p < 0.05). The incidence of litters with 1 or 2 incompletely ossified vertebral sacral arches was increased in the high

- dose group compared to the control group (57.1% vs. 15%; p < 0.05, respectively); this finding was also increased when calculated as the percentage of affected fetuses per litter (16.8% at the high dose vs. 4.1% in controls; p < 0.05). Other observed fetal skeletal variations, which were not considered relatedFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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141 to treatment due to relatively low incidence, comparable incidence among treated and control groups, and/or absence of an increased inciden ce in treated groups compared to the control group, included changes in ossification of the skull, sternebra, phalanges, digits, vertebrae (in addition to effects on sacral arches), and ribs, and variations in the number of ribs.

CONCLUSIONS

The values below are based on initial measured concentrations of test item in the diet, adjusted for test item loss during 3

- 4 day periods in the animal room:

The LOAEL for maternal toxicity in rats was 20.45 mg/kg

- bw/day, based on reduced mean maternal body weight and body weight gain, reduced mean food consumption, decreases in mean carcass weight and mean carcass weight change, and histological changes in the pancreas, including induction of pancreatic acinar cell apoptosis and mononuclear cell inflammation; the NOAEL for maternal toxicity was 7.83 mg/kg

- bw/day

. The LOAEL for developmental toxicity in rats was 20.45 mg/kg

- bw/day , based on reduced mean number of live fetuses per litter, reduced mean number of implantation sites per dam, increased mean number of pre

- implantation losses per dam, increased mean percentage of male fetuses per litter, decreased male and female fetal body weights, decreased placenta weight for female fetuses, and skeletal variations in fetuses, including an increased incidence of unoss ified or incompletely ossified hindlimb metatarsals and incomplete ossification of vertebral sacral arches; the NOAEL for developmental toxicity was 7.83 mg/kg

- bw/day.

STUDY RELIABILITY

[2] Reliable with restrictions. The following deficiencies were not ed: (1) Loss of test substance from feed occurred during the 3

- 4 day intervals between feeding. Although dose calculations were adjusted for estimated loss of test substance, this added variable resulted in an increased degree of uncertainty in actual dos e levels. (2) A reduction in feed consumption was observed at the high dose; it is unclear if other effects at the high dose are all primary toxicological effects or if some of these effects are secondary to reduced feed consumption that may be caused by reduced palatability.

REFERENCES

Theuns

- Van Vliet, J.G., and Otto, M. 2014. An oral prenatal developmental toxicity study with diethylmethylbenzene diamine (DETDA) in rats. Performing Laboratory: TNO Triskelion, The Netherlands. TNO Triskelion Study No. 20193. Sponsors: Albermarle (B elgium), Bayer MaterialScience AG (Germany), and Lonza AG (Switzerland).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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142 E . SUPPLEMENTAL DATA (NON

- SIDS ENDPOINTS)

E

- 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as T1363 and DETA in the study report.

Remarks: Purity not specified; 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

Concentration s tested: 0, 0.7, 7 and 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 untre ated 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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143 implantation losses, as measured by reduction in the number of total implants in test groups as compared with control females. ” [Albemarle Corporation 20 11, p. 26]

“ 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, t he 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 an alyzed: 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 o f 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. [Albemarle Corporation 2011, p. 26]

RESULTS

G enotoxicity 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 mor e dead implants. ” [Albemarle Corporation 2011, p. 27]

“ 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 statistica lly significant dose response was observed. ” [Albemarle Corporation 2011, p. 27]

CONCLUSIONS

“ It was conclude [ d ] DETDA exhibited little or no mutagenic activity in the dominant lethal test. ” [Albemarle Corporation 2011, p. 27]

STUDY RELIABILITY

[3] Not reliable. Purity and composition of the test substance, t est 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 guide line for the Rodent Dominant Lethal Assay, that the highest dose produced signs of toxicity (e.g. slightly reduced fertility or slightly reduced body weight).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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144 REFERENCE S

Schechtman, L., Putman, D., Pannar, A., and Sinsky, P. 1979. Activity of T1363 (ot herwise 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 summarized in Albemarle Corporation

, 2011. ]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes is taken directly from Albemarle Corporation ( 2011)

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145 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, CASRN 68479

- 98

- 1; R eferred to as diethyltoluenediamine (DETDA) in the study report.

Remarks: Purity not specified; 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

- A cetoamido fluor e ne (2AAF) at a concentration of 10

- 6 M, were evaluated concurrently. ” [ Johnson and Mulholland 1984, p. 1]

Isolation of Adult Rat Hepatocytes: “ The protocol for [ isolating adult rat hepatocytes ] is a modification of the method described by Williams (1978). ” [ Johnson and Mulholland 1984, p. 5]

Perfusion Media:

“ Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029. ” [ Johnson and Mulholland 1984, p. 5]

“ Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029. ” [ Johnson and Mulholland 1984, p. 5]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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146 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. ” [ Johnson and Mulhol land 1984, p. 5]

Cell harvesting: “ The liver is excised and the hepatocytes are harvested according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1984, p. 5]

Dosage Selection: “ The highest dose will be 5% solution or the highest soluble conce ntration of the test article in the appropriate solvent. The remaining nine doses will be half

- log dilutions from the 5% solutions. ” [ Johnson and Mulholland 1984, p. 5]

Test Protocol: “ Hepatocytes are treated with the test article, fixed onto microscope cover slides, stained, dipped, and developed according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1984, p. 5]

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 i f 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 c ounts greater than zero. ” [ Johnson and Mulholland 1984, p. 5]

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 determi ned by taking a nuclear count and the average of three adjacent cytoplasmic counts. ” [ Johnson and Mulholland 1984, p. 1]

“ 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. ” [ Johnson and Mulholla nd 1984, p. 6]

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): Slight cytotoxicity was observed at 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. ” [ Johnson and Mulholland 1984, p. 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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147 CONCLUSIONS

“ DETDA was not genetically active in the Rat Hepatocyte Primary Culture/DNA Repair Test according to Protocol

– ETTOX 028. ” [ Johnson and Mulholland 1984, p. 6]

STUDY RELIABILITY

[2] Reliable with restrictions. Purity and composition of test substance w ere not provided. Additionally, the study author did not provide analytical data to verify actual concentrations used in t he 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.

Text in quotes is taken directly from Johnson and Mulholland (1984).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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148 Study 3 : DNA repair test in primary rat hepatocytes ( Johnson and Mulholland, 1986 a )

Title: Rat hepatocyte primary culture/DNA repair test

TEST SUBSTANCE

Identity: Benzenediamine, ar,ar

- diethyl

- ar

- methyl, CASRN 68479

- 98

- 1; Synonym: diethyltoluenediamine (DETDA) ; Referred to as DETDA

- OBG in the study report.

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 toluenediamine (as cited in Mulholland, 1986)

.

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

- Acetoamidofluor e ne (2AAF) at a concentration of 10

- 6 M, were evaluated concurrently. ” [ Johnson and Mulholland 1986 a , p. 5]

Isolation of Adult Rat Hepatocytes: “ The protocol [ for isolating adult rat hepatocytes ] is a modification of the method described by Williams (1978). ” [ Johnson and Mulholland 1986 a , p. 4]

Perfusion Media:

“ Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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149 Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029. ” [ Johnson and Mulholland 1986 a , p. 4]

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. ” [ Johnson and Mulholland 1986 a , p. 4]

Cell harvesting: “ The liver is excised and the hepatocytes are harvested according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1986 a , p. 4]

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. ” [ Johnson and Mulholland 1986 a , p. 4]

Test Protocol: “ Hepatocytes are treated with the test article, fixed onto microscope coverslides, stained, dipped, and developed according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1986 a , p. 4]

Data Quantification: “ Unscheduled DNA repair synthesis, evidenced by a net increase in black silver gr ains 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. ” [ Johnson and Mulholland 1986 a , p. 4]

RESULTS

“ The highest dose scored for DETDA

- OBG 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. ” [ Johnson and Mulholland 1986 a , p. 5]

“ 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 bet ween 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 analysi s was done to compare treated cells to untreated controls. ” [ Johnson and Mulholland 1986 a , p. 5]

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 10, 50, 100, 500 and 1000 μg/wellFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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150 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. ” [ Johnson and Mulholland 1986 a , p. 5]

CONCLUSIONS

“ DETDA

- OBG was not genetically active in the R at H epatocyte P rimary C ulture/DNA R epair T est according to Protocol

– ETTOX 028. ” [ Johnson and Mulholland 1986 a , p. 5]

STUDY RELIABILITY

[2] Reliable with restrictions. The study author did not provide analytical data to verify actual concentrations used i n 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.

Text in quotes is taken directly from Johnson and Mulholland (1986 a ).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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151 Study 4 : DNA repair test in primary rat hepatocytes ( Johnson and Mulholland, 1986 b )

Title: Rat hepatocyte primary culture/DNA repair test

TEST SUBSTANCE

Identity: 3,5

- Diethyltoluene

- 2,6

- diamine, CASRN 2095

- 01

- 4 ( 2,6

- DE TDA isomer of CASRN 68479

- 98

- 1) ; Referred to as 2,6

- DETDA #2 in the study report

.

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 (as cited in Mulholland, 1986)

.

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 a s 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 unt reated control, and 2

- Acetoamido fluorine (2AAF) at a concentration of 10

- 6 M, were evaluated concurrently. ” [ Johnson and Mulholland 1986b, p. 5]

Isolation of Adult Rat Hepatocytes: “ The protocol [ for isolating adult rat hepatocytes ] is a modification of the method described by Williams (1978). ” [ Johnson and Mulholland 1986b, p. 4]

Perfusion Media:

“ Medium A. 0.5 mM EGTA prepared as described in SOP # ETTOX 029.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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152 Medium B. Collagenase, 100 units/mL prepared as described in SOP # ETTOX 029. ” [ Johnson and Mulholland 1986b, p. 4]

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. ” [ Johnson and Mulholland 1986b, p. 4]

Cell harvesting: “ The liver is excised and the hepatocytes are harvested according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1986b, p. 4]

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. ” [ Johnson and Mulholland 1986b, p. 4]

Test Protocol: “ Hepatocytes are treated with the test article, fixed onto microscope coverslides, stained, dipped, and developed according to SOP # ETTOX 029. ” [ Johnson and Mulholland 1986b, p. 4]

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 compoun d 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 ove r background of cells with net nuclear counts greater than zero. ” [ Johnson and Mulholland 1986b, p. 4]

RESULTS

“ The highest dose scored for 2,6

- DETDA #2 was 5 μg/well. Unscheduled DNA repair synthesis was quantified by a net nuclear increase of black si lver grains for 25 cells/slide. This value was determined by taking a nuclear count and the average of three adjacent cytoplasmic counts. ” [ Johnson and Mulholland 1986b, p. 5]

“ 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 dif ference 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. ” [ J ohnson and Mulholland 1986b, p. 5]

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): 10, 50, 100, 500 and 1000 μg/wellFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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153 Results remarks: “ The vehicle and positive control net nuclear grain counts were acceptable. Color and appe arance of the test article did not change from the time of receipt to the time of

use. ” [ Johnson and Mulholland 1986b, p. 5]

CONCLUSIONS

“ 2,6

- DETDA #2 was not genetically active in the Rat Hepatocyte Primary Culture/DNA Repair Test according to Protocol

- Ettox 028. ” [ Johnson and Mulholland 1986b, p. 5]

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. a nd Mulholland, A. 1986 b . 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.

Mulholland, A. 198 6. Recent Genetic Toxicology DETDA Results. Inter

- Office Memo. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Johnson and Mulholland (1986b).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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154 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, CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as “DETA” and identified as “Diethyltoluenediamine” in the study report.

Remarks: Purity not specified; 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:

Repair Test Without Metabolic A ctivation:

“ 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. ” [ Schechtman et al . 1978, p. 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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155 “ Liquid samples of the stock solution are tested at 20, 10 and 2 μl per plate. ” [ Schechtman et al

. 1978, p. 6]

“ Plates are made for each control and each concent ration 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

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 50 0 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. ” [ Schechtman et al . 1978, p. 7]

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. ” [ Schechtman et al . 1978, p. 7]

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 MgCl 2 , 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. ” [ Schechtman et al . 1978, p. 7]

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

. ..” [ Schechtman et al . 1978, p. 7]

“ Prior to use in the assay, each S

- 9 preparati on 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 fo r 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. ” [ Schechtman et al . 1978, pp. 7

- 8]

5. The repair test with metabolic activation

– “ The assay is the same as that described [above]...

, except 0.5 ml of S

- 9 mix is added to the overlay agar immediately before it is m ixed and poured.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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156 Plates are incubated and zones of inhibition are measured as noted above. ” [ Schechtman et al

. 1978, p. 8]

“ 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. ” [ Schechtman et al . 1978, p. 8]

“ Chloramphenicol at 30 μg/plate serves as the negative control. Diethylnitrosamine (DEN) at 10 μl/plate serves as the positive control. ” [ Schechtman et al . 1978, p. 8]

RESULTS

“ Before an agent is reported to be either active or inactive in the DNA Repair test with E

. coli

, th e following criteria must be met:

All sterility controls, including test compound and S

- 9, must be negative for bacterial growth. S

- 9 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 r epeated on a different day. ” [ Schechtman et al . 1978, p. 9]

“ The tests reported upon below met each of the above mentioned criteria and the test agent was evaluated accordingly. ” [ Schechtman et al . 1978, p. 9]

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 concen tration of 100 μl of S

- 9 per ml of S

- 9 mix. ” [ Schechtman et al . 1978, p. 9]

“ 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. ” [ Schechtman et al . 1978, p. 9]

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. ” [ Schechtman et al . 1978, p. 9]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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157 CONCLUSIONS

“ Under the conditions of this test, DETA 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

. ” [ Schechtman et al . 1978, p. 12]

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 Inhibiti on of Repair Deficient and Repair Competent Strains of Escherichia coli : DNA Repair. Performing Laboratory: Microbiological Associates. Bethesda, MD. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Schechtman et al . ( 1978).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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158 Study 6 : In vitro mammalian cell transformation assay ( Schechtman et al., 1978)

Title: Activity of DETA in i n vitro mammalian cell transformation assay

TEST SUBSTANCE

Identity: Benzenediamine, ar,ar

- diethyl

- ar

- methyl, CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as “DETA” and identified as “Diethyltoluenediamine” in the study report.

Remarks: Purity not specified; 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 prese nce 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 N

methyl

- 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 o r dimethylsulfoxide (DMSO), the solvent vehicle, +/

- S

- 9 serves as the negative control.

“ Exponentially growing BALB/3T3 C1. A31 cells were cultured in 75 cm 2 cell culture flasks and collected by trypsinization. Cells were seeded in triplicate dishes per condition at densities ofFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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159 250 and 500 cells per 60 mm dish for determinations of the relative cytotoxic effects of each treatment and 1 x 10 4 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. ” [Schechtman et al . 1978, p. 6]

“ 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 an d 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. ” [Schechtman et al . 1978 , p. 6]

“ 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 plati ng 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

.. . ” [Schechtman et al . 197 8, p. 6]

“ 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

... The tran sformation 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 ...” [Schechtman et al . 1978, p. 7]

“ A preliminary cytotoxicity test was conducted to determine the dose range to be employed in the transformation assay. Whenever possible, the approximate LD 50 concentration of the test chemicals plus 1 dose above and 1 dose below the LD 50 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 M NNG was used as the positive control. ” [Schechtman et al . 1978, p. 7]

The following text is taken directly from Appendices A and B of the study report:

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 con dition, 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% CO 2 + air.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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160 2. Twenty

- four hours later, 2X concentrations of test chemical (6 doses) or solvent v ehicle (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. Ho wever, if desirable, the highest dose to be tested will be determined by the limits of solubility of the test agent.

4. After a 24 hr exposure period, the treatment medium is withdrawn; cells are washed once with Hanks’ balanced salt solutions (pH 7.0) an d 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 LD 50 is determined from the relative survival data. The LD 50 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 10 4 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% CO 2 + 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 LD 50 , one dose above and one dose below the LD 50 . (The LD 50 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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161 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 contact

inhibition of cell growth and division, resulting in a randomly oriented piled

- up array of cells. Cellul ar constituents of such transformed foci grow into colonies in semi

- solid agar (whereas

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 i n 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. Repr esentative 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 DETA on the BALB/3T3 C1. A31 cells, “ a dose response was not observed due to the extreme toxicity of DETA to the target cells. The test agent was 100% cytotoxic at all of the concentrations tested alth ough a few single cells were observed at the 1:1000 dilution; however, no colony formation was observed. Compound DETA precipitated heavily at all of the concentrations tested. ” [Schechtman et al . 1978, p. 8]

In the additional preliminary study utilizing revised dilutions to determine the cytotoxic effects of DETA on BALB/3T3 C1. A31 cells and to establish the approximate LD 50 of the test compound to the target cells, “ DETA was observed to be 100% cytotoxic at the 1:1000, 1:2000 and 1:3000 dilutions. Alt hough 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. ” [Schechtman et al . 1978, p. 8]

In the cytotoxicity study of the compound DETA which accompanied the transformation assay, a dose response was observed. “ Although DETA 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 LD 40 was found to be at the 1:30000 dilution of compound DETA. ” [Schechtman et al . 1978, p . 8]

In the cell transformation test in the absence of exogenous metabolic activation, DETA was ineffective in producing Type III foci at all of the concentrations tested. “ The negative control condition (medium control) exhibited no spontaneously transf ormed foci whereas, the positive control condition (MNNG) produced 4 morphologically transformed foci (transformation frequency of 6.0 x 10

- 4 ). ” [Schechtman et al . 1978, p. 8]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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162 Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 1:30000 (dilutions of test agent in reaction mixture)

Results remarks: None specified

CONCLUSIONS

“ Based on the results and discussion the following conclusions were made: (1) DETA was found to be extremely cytotoxic to the BALB/3T3 C1. A31 cells. (2) DETA 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 DETA was incapable of inducing morphologica l transformants (type III foci) at any of the concentrations tested. ” [Schechtman et al . 1978, p. 13]

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.

Text in quotes is taken directly from Schechtman et al . (1978).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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163 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 ; Referred to as “DETA” , “Diethyltolueneamine”, and T1365A in the study report.

Remarks: Purity not specified ; 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 10 4 , 1:1.5 x 10 4 and 1:2 x 10 4 (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 prese nce 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 N

methyl

- 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 o r dimethylsulfoxide (DMSO), the solvent vehicle, +/

- S

- 9 serves as the negative control.

“ Exponentially growing BALB/3T3 C1. A31 cells were cultured in 75 cm 2 cell culture flasks and collected by trypsinization into 50 cc conical tubes for treatment in su spension at 27°C for 2Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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164 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) ” [Schechtman et al . 1979, p. 6]

“ 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. ” [Schechtman et al . 1979, p. 6]

“ 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 10 4 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. ” [Schechtman et al . 1979, p. 7]

“ 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 colon ies in order to determine the relative plating efficiencies (RPE) according to the protocol of Schectman and Kouri ...” [Schechtman et al . 1979, p. 7]

“ 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 ...” [Schechtman et al

. 1979, p. 7]

“ 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. ” [Schechtman et al . 1979, p. 7]

“ A preliminary cytotoxicity study was conducted to determine the dose range to be employed in the transformation assay.

.. Whenever possible, the approximate LD 50 concentration of the test chemical plus one dose above and one dose below the LD 50 were those used in the
tra nsformation assay. ” [Schechtman et al . 1979, p. 7]

The following text is taken directly from Appendices A

- C of the study report:

APPENDIX A: Standard Protocol for the Determination of the Cytotoxic Effects of Test Chemicals to BALB/3T3 C1. A31 Mouse Ce lls in the Presence of Exogenous Metabolic Activation: Suspension AssayFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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1 65 1. Two

- 3 x 10 6 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 disca rded 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 Final concentrations of the test chemical employed are those used in the accompanying mutation/transformation assay.

3. The tubes containing ea ch reaction mixture are incubated for 2 hr at 37 C in a humidified atmosphere of 5% CO 2 + 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 washin g 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 day s 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 se eded), and/or relative colony

- forming efficiency (RCE) (i.e. number of colonies in chemically treated dishes relative to the number of colonies in solvent

treated controls).

APPENDIX B: Standard Protocol for the Morphological Transformation of BALB/3T3 C 1. A31 Mouse Cells in the Presence of Exogenous Metabolic Activation: Suspension Assay

1. Two

- 3 x 10 6 logarithmically growing BALB/3T3 C1. A31 cells from sub

- confluent stock cultures are collected into 50 cc capacity conical centrifuge tubes (two tubes pe r 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 cof actor pool) +/

- test chemical. The test conditions include:Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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166 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 shal l be based upon the results obtained in a preliminary cytotoxicity assay, and shall include, whenever possible, the approximately LD 50 , one dose above and one dose below the LD 50 . 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 2hr at 37°C in a humidified atmosphere of 5% CO 2 + 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 10 4 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 tra nsformation 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 di vision, 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 o f 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 Effe cts 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) containingFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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167 10% heat

- inactivated fetal bovine serum, and incubated at 37°C in a humidified atmosphere of 5% CO 2 + air.

2. Twenty

- four hours later, 2X concentrations of test chemical (6 doses) or solvent vehicle (negativ e 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 desirabl e, 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 solvent

treated controls).

7. The approximate LD 50 is determined from the relative survival data. The LD 50 is that dose of the test chemical which is lethal to 50% of the target cells.

RESULTS

Under the conditions of the preliminary study, “ 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. ” [Schechtman et al . 1979, p. 8]

In an additional preliminary study utilizing revised dilutions, “ T1365A was observed to be 100% cytotoxic at the 1:1 x 10 3 , 1:2 x 10 3 and 1:3 x 10 3 dilutions. Although cell survival and colony formation was observed at the 1 : 5 x 10 3 dilution, the relative survival of the target cells at this concentration was judged to be too low to be used in the transformation assay. ” [Schechtman et al . 1979, p. 8]

“ These results necessitated an additional c ytotoxicity assay which would serve to estimate the approximate LD 50 of the test compound in the presence of exogenous metabolic activation. ” [Schechtman et al . 1979, p. 8] 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 t a rget cells expressed in terms of (%) relative survival after treatment could be established. Under these conditions, compound T1365A was again observed to be extremely cytotoxic, even at a 1:1 x 10 4 dilution (Relative Survival ≤10%). A dramatic decrease in the level of toxicity wasFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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168 observed at dilutions ≥1:1.5 x 10 4 . The observed cellular response made it difficult to establish an LD 50 for test agent T1365A. Therefore, the transformation assay (and its accompanying cytotoxicity assay) was performed usin g these same doses in order to bracket the LD 50 range. ” [Schechtman et al . 1979, p. 8]

A quantitative cytotoxicity study accompanied the transformation assay. “ Under the conditions of this assay as performed... , an approximate LD 6 0 was observed at the 1:1 x 10 4 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. ” [Schechtman et al . 1979, p. 8]

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) foc i (transformation frequency of 1.6 x 10

- 4 ), whereas the negative control condition (S

- 9 + buffer) failed to induce spontaneous transformants. ” [Schechtman et al . 1979, p. 8]

The transformation frequencies at 1:1 x 10 4 , 1:1.5 x 10 4 , and 1:2 x 10 4 were < 0.55 x 10 4 , <0.26 x 10 4 and <0.23 x 10 4 , respectively.

Genotoxicity result: Negative

Cytotoxic concentration (if applicable): ≥ 1:2 x 10 4 (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 p eriod 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 e mployed, the test chemical was ineffective in inducing morphological transformation (Type III foci) in the BALB/3T3 Clone 31 cell system at all concentrations employed. ” [Schechtman et al . 1979, p. 14]

STUDY RELIABILITY

[3] Not reliable. Important detai ls 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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169 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 Activatio n. Study No. T1365A. Performing Laboratory: Microbiological Associates, Bethesda, MD. Sponsor: Ethyl Corporation, Baton Rouge, LA.

Text in quotes is taken directly from Schechtman et al . (1979).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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170 E

- 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, CASRN 68479

- 98

- 1; Referred to as Diethyl Toluene Diamine (DETDA) in the study report.

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

Exp osure duration: Daily, 7 days a week for 24 months (731 days)

Doses/concentrations tested: 0 (untreated control), 10, 35, and 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, hematolog y 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.,Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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171 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. ” [Margitich and Ciofalo 1992, p. 7]

Test method/conditions rem arks: “ The test article, Diethyl Toluene Diamine (DETDA), 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 di et 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 n ecropsy 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 an d 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. ” [Margitich and Ciofalo 1992, p. 1]

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 aden omas 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 maj ority 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. ” [Margitich and Ciofalo 1992, p. 1]

“ 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 remainder 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 me an body weightFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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172 in the mid dose males was seen beginning at about month 15 and continuing to the conclusion of the study. No effect, statistical or otherwise, was found on the body weight of low dose males compared to control males. ” [Margitich and Ciofalo 1992, p. 1]

“ 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 lo wer 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 mid

dose female body weight were fo und. 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. ” [Margitich and Ciofalo 1992, pp. 1

- 2]

“ 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 d aily food consumption. ” [Margitich and Ciofalo 1992, p. 2]

“ 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. ” [ Margitich and Ciofalo 1992, p. 2]

“ 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 5 0% 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. ” [Margitich and C iofalo 1992, p. 2]

“ 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 articl e. ” [Margitich and Ciofalo 1992, p. 2]

“ 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 adeno mas 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 ofFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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173 the mammary glands of mid and high dose females. Hepatic proliferative lesions (b asophilic 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 multifo cal necrosis). Basophilic foci in the liver of 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 ac companied 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 arti cle. 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 ne arly 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 inc idental lesions were present in both the control and DETDA treated rats. ” [Margitich and Ciofalo 1992, pp. 2

- 3]

“ 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 clear no effect level for liver and thyroid tumors can be established at 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. ” [Margitich and Ciofalo 1992, pp. 4

- 5]

“ The results of this study should be evaluated in conjunction with the earlier DETDA studies conducted in this 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 cel l 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 eff ects 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 toxic ity was not seen. ” [Margitich and Ciofalo 1992, p. 3]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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174 “ The greater DETDA sensitivity of male, compared to female, rats was also observed in the two year 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. ” [Margitich and Ciofalo 1992, p. 4]

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 consum ption 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
pancr eatic toxicity in the male rat for two years was 35 ppm; for females the no effect level was 70 ppm. ” [Margitich and Ciofalo 1992, p. 4]

“ 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. ” [Margitich and Ciofalo 1992, p. 4] 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 [see review of this study (Naismith and Matthews 1987) in a separate sum mary 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. ” [Margitich and Ciofalo 1992, p. 4]

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 S

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.

Text in quotes is taken directly from Margitich and Ciofalo (1992).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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175 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, CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA) ; Referred to as “diethyltolamine” and “ diethyltoluenediamine ” in the study report.

Remarks: 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 7 4 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 and 12 mg/kg

- bw/day; Diet study

– 0 (control), 80 and 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 groupsFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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176 per gender are compared to each other by means of generalized SAVAGE test. ” [ Steinhoff 1984

, p. 15]

“ 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. ” [ Steinhoff 1984 , p. 15]

“ 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. ” [ Steinhoff 1984 , p. 15]

“ The possibility of a relationship between the tumor findings and the dosage is tested on the basis of recommendati ons 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. ” [ Steinhoff 1984 , p. 15]

“ 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 d ie 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 facto rs applied to the testing of these hypotheses are 0, 1 and 2 (increasing with dose). ” [ Steinhoff 1984 , p. 16]

“ 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. ” [ Steinhoff 1984 , p. 16]

“ The evaluation of the data is carr ied 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 previousl y in the Department of Biometrics, EP

- FE (Prevalence rates by Dr. Dycka). ” [ Steinhoff 1984 , p. 16]

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. ” [ Steinh off 1984 , p. 17]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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177 “ The number of the 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 wi th a positive tumor finding and according to the dignity of the findings. ” [ Steinhoff 1984 , p. 17]

“ 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 histologi c ally benig n, histologically malignant and (positively identified) tumors. The cumulative numbers of animals with a particular tumo u r are tabulated at weekly intervals. ” [ Steinhoff 1984 , p. 17]

“ 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 di stinguished 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 comp ared to the two groups receiving substance. This in turn determines whether tests for inhomogeneity or trend should be made. ” [ Steinhoff 1984 , pp. 17

- 18]

“ The test is therefore carried out in two stages: firstly, the hypothesis is tested

– formally in th e 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 dif ferent. 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 factor s applied to the testing of these hypotheses are 0, 1 and 2 (increasing with dose). ” [ Steinhoff 1984 , p. 18]

“ The generalized evaluation by the prevalence method leads to only 2 time interval classes in the female animals when the time course is taken in to consideration. Since only one animal dies in the first time interval the analysis is ended at this point. ” [ Steinhoff 1984 , p. 18]

“ 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). ” [ Steinhoff 1984 , p. 18]

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 LD 50 ). The substance was mixed with arachis oil and administered by gavage on a single occasion to groups of 10 female S prague

- 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 ...” [ Steinhoff 1984 , p. 8]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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178 “ The 90

- day study was based on four groups of 10 Wistar rats (5 males and 5 females ) aged about 8 months. In this preliminary study the DETDA (Batch 74) was mixed in with the powdered feed i n concentrations of 0, 100, 300 and 900 mg/kg feed (ppm). Th e 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. ” [ Steinhoff 1984 , p. 8]

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 feedi ng 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 w ere 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 y ears (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 st udy 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. ” [Albemarle Corporation 2011, p. 22]

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. ” [ Steinhoff 1984 , p. 19] The LD 50 (confidence interval p ≤ 0.05) was 598 (568

- 653) mg/kg body weight.

“ As in the acute toxicity s tudy, 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 particul ar. 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 th at 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 12 th week, without any abnormal findings. ” [ Steinhoff 1984 , p. 19]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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179 “ 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. ” [ Steinhoff 1984 , pp. 19

- 20]

“ The auto psies of the animals in the highest concentration group (900 ppm) performed at the end of the study did not yield any particular findings. ” [ Steinhoff 1984 , p. 20]

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 a t the upper doses were more severely affected than females. ” [Albemarle Corporation 2011, p. 22]

“ No evidence of carcinogenicity was found. When results by the two methods of administration were considered together, a dose

- dependent increase in the num ber 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. ” [Albemarle Corporation 2011, pp. 22

- 23]

CONCLUSIONS

The report concluded that no evidence of carcinogenicity was found de spite the fact that the test animals were treated with the maximum tolerated to toxic doses.

STUDY RELIABILITY

[2] Reliable with restrictions. A complete description of the test substance composition was not provided. Test method/guideline and GLP compliance w ere not specified

REFERENCE S

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.

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes is taken directly from Steinhoff (1984) and Albe marle Corporation (2011)

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180 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, CASRN 68479

- 98

- 1; Synonym: Diethyltolenediamine (DETDA) ; Referred to as Diethyltolamine Technical Experimental Product DD 1443 batch 1 in the study report.

Remarks: 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 complia nt?: 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 LD 50 was determined after single subcut aneous doses to female specifically bred Winkelmann Wistar rats (W 64), weighing approximately 140 g. These were undiluted injections of the preparation. The LD 50 determined was 368 (338

- 400) mg/kg (p = 0.05). The surviving rats showed a continuous dege neration of general condition during the four

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181 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 another strain [ 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 al lowed to survive over their natural lifespan. The dose used in the study was based on the LD 50 . The LD 50 via subcutaneously injection was determined to be 368 mg/kg, and doses lower than this were selected for the carcinogenicity study. ” [Albemarle Corpo ration 2011, p. 21]

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 deve loped: a cystic papilloma and two subcutaneous fibromas. ” [ Steinhoff 1972 , p. 6]

“ 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 tumo r 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. ” [ Steinhoff 1972 , p. 6]

“ 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 polymorphou s

- cells carcinoma of the brain. Besides these, a benign tumor of the thymus developed. ” [ Steinhoff 1972 , p. 7]

“ 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. ” [ Steinhoff 1972 , p. 7]

“ 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, on e uterine polyp, and a lipoma on the thorax. ” [ Steinhoff 1972 , p. 7]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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182 “ 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 observe d in the control group: 3 fibroadenomas of the breast and 2 uterine polyps. ” [ Steinhoff 1972 , p. 7]

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 dosa ge, 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). ” [ Steinhoff 1972 , p. 7]

“ 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 t he 50 rats (25 males, 25 females). ” [ Steinhoff 1972 , p. 8]

“ 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). ” [ Steinhoff 1972 , p. 8]

“ 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. ” [ Steinhoff 1972 , p. 8]

“ All together, then, among the 50 rats treated with techni cal 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. ” [ Steinhoff 1972 , p. 8]

“ There is, th en, 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 whic h 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. ” [ Steinhoff 197 2 , p. 8]

“ 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 survivalFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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183 times. It is also true that these animals were exposed to o verall 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 c learly reduced survival time. ” [ Steinhoff 1972 , p. 8]

STUDY RELIABILITY

[2] Reliable with restrictions. Several details were missing from the study including a complete description of the composition of the test substance, test method/guideline followed, GLP compliance and statistical methods used.

REFERENCE S

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. Repor t No. 3709. Performing Laboratory: Bayer AG Toxicological Institute. Sponsor: Bayer AG, Germany.

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes is taken directly from Steinhoff (1972) and Albemarle Corporation (2011)

.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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184 E

- 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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 we re 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. ” [Albemarle Corporation 2011, p. 10]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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185 RESULTS

Endpoint value(s): Slightly irritating. The primary dermal irritation index based on erythema, edema and eschar formation was 1.7.

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 S

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. S ponsor: Ethyl Corporation, Baton Rouge, LA. [As summarized in Albemarle Corporation , 2011.]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.

Text in quotes is taken directly from Albemarle Corporation (2 011).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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186 E

- 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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187 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. Perfo r ming Laboratory: Bio/dynamics Inc. S ponsor: Ethyl Corporation, Baton Rouge, LA. [As summarized in Albemarle Corporation, 2011.]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of September 12 , 2014.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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188 E

- 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

- , CASRN 68479

- 98

- 1; Synonym: Diethyltoluenediamine (DETDA)

Remarks: Purity not specified; 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 10 th injection, a challenge injection of 0.05 mL was administered. Sites were scored at 24 and 48 hrs after the last injec tion. An additional 4 guinea pigs were treated as described above with the positive control, 0.1% dinitrochlorobenzene in ethylene dichloride. ” [Albemarle Corporation 2011, p. 10]

Test condition: Not indicated

RESULTS

Endpoint value(s): Not sensitizing.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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189 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. S ponsor: Ethyl Corporation, Baton Rouge, LA. [As summarized in Albemarle Corporation, 2011.]

Albemarle Corporation. 2011. Letter from Kim Boudreaux, Regulatory Advisor, Albemarle Corporation to Document Control Office, Office of Pollution Prevention and Toxics (OPPT) , US EPA (letter dated May 4, 2011). Submission available online at http://www.regulations.gov/ #!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0632 as of Septem ber 12 , 2014.

Text in quotes is taken directly from Albemarle Corporation (2011).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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190 E

- 6. Toxicokinetic Studies

Study 1: Toxicokinetic study in Fischer 344 rats ( Hainje and El

- hawari, 1982)

Title: Absorption, distribution, elimination, and bioretention of 14 C

- labeled diethyltoluenediamine following repeated oral dosing in rats

TEST SUBSTANCE

Identity: Benzenediamine, ar,ar

- diethyl

- ar

- methyl, CASRN 68479

- 98

- 1; Referred to as Diethyl Toluene Diamine (DETDA) in the study report.

Remarks: Purity: > 99%; 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
two

- tailed Student's t test. Significant differences were indicated when p < 0.05. ” [ Hainje and El

- hawari 1982

, p. 8]

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 oral doses (50 mg/kg [

- bw ] ) of the 14 C

- labeledFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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191 compou nd ( 14 C

- DETDA). Control rats received nine daily doses of the vehicle (0.05 N HCl) followed by the tenth dose of 14 C

- 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. ” [ Hainje and El

- hawari 1982

, p. 1]

“ 14 C

- DETDA was administered [ to 6 rats ] by gavage at a dose level of 50 mg/kg (

~ 1/10 of the LD 50 ) as specified by the sponsor. A limited preliminary study was also performed in two rats treated with a 10 mg/kg dose of 14 C

- DETDA. The dosing solutions were prepared by dissolving the appropriate amounts of the nonlabeled and 14 C

- labeled DETDA in a volume of 5 ml/kg of 0.05 N hydrochloric acid. Fresh dosing solutions were prepared for the pre liminary 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. ” [ Hainje and El

- hawari 1982

, p. 5]

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
14 C

- DETDA. 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 radioac tivity 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 14 C

- 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). ” [ Hainje and El

- hawari 1982

, p. 5]

“ 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. Urin e was collected at 4, 8, 12 and 24 hr, and feces were sampled at 12 and 24 hr, and then the rats were sacrificed. ” [ Hainje and El

hawari 1982

, p. 5]

“ Respiratory elimination of 14

- CO 2 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 CO 2 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. ” [ Hainje and El

- hawari 1982

, p. 6]

“ Urinary and fecal excretion were assessed during a 96

- hr period in one male and one female rat treated with a 50 mg/k g dose, and one male and female treated with a 10 mg/kg dose of 14 C

DETDA. 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, an d then the rats were sacrificed. ” [ Hainje and El

- hawari 1982

, p. 6]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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192 Definitive studies: “ A total of 24 rats (12 males and 12 fe males) 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
14 C

- DETDA or 9 daily doses of the vehicle followed by a tenth dose of 14 C

- DETDA. D aily 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 fem ales from each group were used for 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

... ” [ Hainje and El

- hawari 1982

, p. 6]

“ 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 ...” [ Hainje and El

- hawari 1982

, p. 7]

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 RB Cs 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. ” [ Hainje and El

- hawari 1982

, p. 7]

Sample Preparation and Analysis: “ Aliquots (50 to 250 μ l ) of wh o le 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 14 C content. Blood components, tissues, and fecal samples were combusted in a Packard Tricarb Sample Oxi dizer (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 Counti ng Solution (PCS, Amersham). ” [ Hainje and El

- hawari 1982

, p. 7]

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 aut omatic 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 reassa yed in duplicate except when the sample was no longer available or whenFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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193 radioactivity counts were low and nonsignificant, i.e., less than two times the background, 30 cpm (counts per minute). ” [ Hainje and El

- hawari 1982

, p. 7]

Data Processing and Analysis : “Carbon

- 14 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 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. ” [ Hainje and El

- hawari 1982

, p. 8]

“ 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. ” [ Hainje and El

- hawari 1982

, p. 8]

RESULTS

Preliminary Study: “ [ A ] single oral dose of 14 C

- 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 highes t in thyroids, followed by liver, kidneys, and adrenals. Male rats demonstrated higher levels of 14 C in thyroids and liver than females. ” [ Hainje and El

- hawari 1982

, p. 1]

Definitive Study: “ Following the last dose, rats treated with 10 doses of 14 C

- DETD A displayed significantly (up to 5

- fold) higher levels of radioactivity in blood than rats treated with one dose. The females demonstrated higher 14 C

- 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 14 C was greater in rats treated with 10 doses as compared to those receiving 1 dose of 14 C

- DETDA

. Female rat s of both treatment groups eliminated higher amounts of 14 C in urine than their male counterparts. ” [ Hainje and El

- hawari 1982

, p. 1]

“ At 24 and 96 hr after the last doses, the concentration of ra d ioactivity in tissues was highest in thyroids, followed by liver, kidneys, and adrenals. 14 C levels in skin were higher than in fat or muscle.

.. Rats treated with 10 doses displayed significantly higher levels of 14 C in most tissues examined than those t reated 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 14 C in liver but higher amounts in blood, RBCs, brain, spleen, and pancrea s. At 96 hr, liver and thyroids of the females contained lower 14 C 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 d ose as compared to those receiving 10 doses of 14 C

- DETDA. ” [ Hainje and El

- hawari 1982

, p. 1]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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194 “ In rats treated with 10 doses of 14 C

- 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 9 6 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 ”

. [ Hainje and El

- hawari 1982

, p. 1]

Results remarks: “ Since control rats (i.e., nontreated with DETDA) were not included in the studies, no at tempt was made to statistically analyze the changes in these [ bodyweight and organ weight ] parameters. ” [ Hainje and El

- hawari 1982

, p. 9]

CONCLUSIONS

“ The results of these studies indicate that 14 C

- labeled diethyltoluenediamine ( 14 C

- 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. Radioactivi ty 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; 14 C levels in liver and thyroids w ere higher in the males. ” [ Hainje and El

- hawari 1982

, p. 14]

“ Treatment of Fischer 344 rats with 10 daily oral doses of the 14 C

- labeled compound resulted in significantly higher blood levels of 14 C compared to those of rats receiving one dose (vehicle

pre treated 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 14 C

- DETDA, the amounts of radioactivity retained in blood and tissue of the latter gr oup 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. ” [ Hainje and El

- hawari 1982

, p. 14]

“ The data obtained from these studies did not indicate an impairment of the excretory functions in rats pretreated with 14 C

- DETDA. Therefore, the retention of radioactivity in blood and tissues of rats receiving the 10 daily doses of the labeled compound suggests that the 14 C level (probably from metabolites) binds to certain blood and tissue components. Definitive explanations of these findings require further assessment of the biotransformation a nd disposition of DETDA at several dose levels and its possible interaction with blood and tissue macromolecules. ” [ Hainje and El

- hawari 1982

, p. 14]

STUDY RELIABILITY

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

.
REFERENCE S

Hainje, M. and El

- hawari, A. 1982. Absorption, Distribution, Elimination, and Bioretention of
14 C

- Labeled Diethyltoluenediamine Following Repeated Oral Dosing to Rats. MRI Project No. 7348

- B. Performing Laboratory: Midwest Research Instit ute, Kansas City, MO. Sponsor: Ethyl Corporation, Baton Rouge, LA.Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

195 Text in quotes is taken directly from Hainje and El

- hawari (1982).Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

196 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, CASRN 68479

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- 1; Referred to as Diethyl Toluene Diamine (DETDA) and Ethacure 100 in the study report.

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 (84 μmol/ml) in water to provide a labeled mixtu re 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 μ mol/kg

- bw

Statistical methods used: None specified

Test method/conditions remarks: Using synt hesized 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
contr ol and 3 treatment, 172

- 186 g) were administered a single dose of vehicle or the commercialFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

197 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 non

- tritiated Ethacure in water (to provide a l abeled mixture with a specific activity of 0.46 μCi/μmol) at 179 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 inc orporated in the 6 day excretion stud y 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). ” [Babin et al . 1996, p p . 74

- 75 ]

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

ov er 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 are a), liver (right caudal lobe), stomach (greater curvature), proximal duodenum, caecum (apex), colon (proximal ascending) and brain (right frontal lobe). ” [Babin et al . 1996, p p . 76

- 77 ]

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 sacrif iced 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, “ o nly 2 of the 3 rats administered Ethacure were quantitatively evalu ated 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. ” [Babin et al . 1996, p. 60]Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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198 “ 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 showe d 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

... w ere 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. ” [Babin et al . 1996, p. 87]

“ 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

- DET D A. 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, fo r nonexcretory organs, with thyroid levels exceeding even that of l iver 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 gleaned 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. ” [Babin et al . 1996, p. 130]

“ 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. ” [Babin et al . 1996, p. xvi]

“ Differences i n 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
met abolite 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. ” [Babin et al . 1996, p. xvi]

“ Analysis of the data from the 24

- hour time point urine samples revealed that 18.2% 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 labeledFinal HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

199 material was composed of the non acid

- labile water soluble fraction. ..” [Babin et al . 1996, p p

. xvi

- xvii ]

“ Relative to Ethacure (80.8% 2,4

- DETDA, 19.2% 2,6

- DETDA), 1 6 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 t o be involved in covalent binding to DNA. ” [Babin et al . 1996, p. xvii]

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 stu dy included test substance purity, method/guideline followed and GLP compliance.

REFERENCE S

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.

Text in quotes is taken directly from Babin et al . (1996)Final HPV2 Test Rule Data Adequacy Review (September 2014) CASRN 68479

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- 1

200 F

. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish Yes Yes No Acute Toxicity to Aquatic Invertebrates Yes Yes N o Toxicity to Aquatic Plants 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

– No 1 Developmental Toxicity Yes Yes No

1 The reproductive toxicity endpoint has been acceptably addressed on the basis that an adequate developmental toxicity study is available and no treatment

- related histopathological effects in reproductive organs were observed at sublethal dietary concentrations of CASRN 68479

- 98

- 1 in adequate 90

- and 28

- day repeated

- dose toxicity studies and a two

- year cancer study

.