Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2014 to 19 September 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Appearance/physical state: Black solid
- Storage conditions: Room temperature, protected from light under nitrogen

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolising system (10 % liver S9 in standard co-factors)

Test concentrations with justification for top dose:

- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 2: 1.5, 5, 15, 50, 150, 500 and 1500 μg/plate

Vehicle / solvent:

Dimethyl sulphoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

Controls 3

Positive controls:

yes

Remarks:

ENNG without S9 mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 µg/plate for WP2uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535

Controls 4

Positive controls:

yes

Remarks:

9AA without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Controls 5

Positive controls:

yes

Remarks:

4NQO without S9 mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98

Controls 6

Positive controls:

yes

Remarks:

2AA with S9 mix

Positive control substance:

other: 2-aminoanthracene

Remarks:

1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA

Controls 7

Positive controls:

yes

Remarks:

BP with S9 mix

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98

Details on test system and experimental conditions:

TESTER STRAINS
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain L T2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TAI 00~ the R-factor plasmid pKMIOI enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains auvrA-DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1408880 10/18) and incubated at 37 °C for approximately I 0 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

TEST ITEM
- The test item was insoluble in sterile distilled water at 50 mg/mL but fully soluble in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period.
- Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
- Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10E-4 microns.

CONTROL ITEMS
- Vehicle, negative (untreated) and positive controls were used in parallel with the test item.
- The vehicle control used was dimethyl sulphoxide.

MICROSOMAL ENZYME FRACTION
- Lot No. PB/PNF S9 27 July 2014 was used in the study.
- The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/ß-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
- The S9 homogenate was produced by homogenizing the liver in a 0.15 M KCl solution (1 g liver to 3 mL KCl) followed by centrifugation at 9000 g.
- The protein content of the resultant supernatant was adjusted to 20 mg/mL.
- Aliquots of the supernatant were frozen and stored at approximately -196 °C.
- Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
- The procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012.

S9 MIX AND AGAR
- The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
- The S9 mix contained S9 (5.0 mL); 1.65 M KCl/0.4 M MgCl2 (1.0 mL); 0.1 M glucose-6-phosphate (2.5 mL); 0.1 M NADP (2.0 mL); 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL); sterile distilled water (14.5 mL).
- A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
- Top agar was prepared using 0.6% Bacto agar (lot number 3218431 04/18) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 37294 09/14 and 37306 09/14).

DOSE SELECTION FOR EXPERIMENT 1
The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
- Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- The appropriate concentration of test item or solvent (0.1 mL) or appropriate positive control (0.1 mL) was added to 2mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test.
- Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the test item formulation and bacterial culture, 0.5 mL of 89-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

DOSE SELECTION FOR EXPERIMENT 2
- The results of Experiment 1 determined the Experiment 2 dose range (1.5, 5, 15, 50, 150, 500 and 1500 μg/plate).
- Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously.

INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
(ii) All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2012 and 2013 are presented in Appendix 2 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E-09 bacteria per mL.
(iv) Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2012 and 2013 are presented in Appendix 2 (attached).
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

Evaluation criteria:

EVALUATION CRITERIA
- There are several criteria for determining a positive result. Any one, or all, of the following can be used to determine the overall result of the study:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(v) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by · an out-of-historical range response (Cariello and Piegorsch, 1996) ).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

MUTATION TEST

- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (attached) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 (attached) and Table 4 and Table 5 for Experiment 2 (attached).

- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2 (attached).

- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate and the toxic limit selected as the maximum dose in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 μg/plate (TA1537 dosed in the absence of S9-mix) and from 500 μg/plate to all of the remaining tester strains dosed in both the presence and absence of S9-mix. A dark grey colouration was noted from 150 μg/plate (second experiment) and a test item precipitate (greasy in appearance) was noted by eye at and above 500 μg/plate. Neither of these observations prevented the scoring of revertant colonies.

- There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in TA98 revertant colony frequency was observed in the presence of S9-mix

at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns.

Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.

- All of the positive control chemicals usedinthe test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

GUIDELINE

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TAlOO andEscherichia colistrainWP2uvrAwere treated with the test item using the Ames plate incorporation method at up toeight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was pre-determined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was

amended, following the results of Experiment 1, and was 1.5 to 1500 μg/plate. Up to seven test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

RESULTS

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate and the toxic limit selected as the maximum dose in the second mutation test. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 μg/plate (TA1537 dosed in the absence of S9-mix) and from 500 μg/plate to all of the remaining tester strains dosed in both the presence and absence of S9-mix. A dark grey colouration was noted from 150 μg/plate (second experiment) and a test item precipitate (greasy in appearance) was noted by eye at and above 500 μg/plate. Neither of these observations prevented the scoring of revertant colonies. There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test. Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2. A small, statistically significant increase in T A98 revertant colony frequency was observed in the presence of S9-mix

at 150 μg/plate in the first mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Further statistically significant increases were also noted, however these responses were accompanied by weakened bacterial background lawns.

Therefore the responses in this instance would be due to additional amino acid being available to the bacteria allowing the cells to undergo several additional cell divisions and presenting as non-revertant colonies.

 

CONCLUSION

The test item was considered to be non-mutagenic under the conditions of this test.