Reaction products of 4,4'-methylenebis(2,6-dimethylaniline) and 4-aminobenzenesulfonic acid with 2,4,6-trichloro-1,3,5-triazine, diazotised and subsequently coupled with N-(2-methoxyphenyl)-3-oxobutanamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: Sokolowski (2006)

Under the conditions of the study the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 March 2006 to 17 March 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 - Environmental Protection Agency.
Eisei No. 127 - Ministry of Health and Welfare.
Heisei 09/10/31 Kikyoku No. 2 - Ministry of International Trade and Industry.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- On the day of the experiment the test material was dissolved in DMSO.

Target gene:

S. typhimurium strains: Histidine
E. coli WP2 uvrA: Tryptophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The bacterial strains TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA were obtained from Trinova Biochem GmbH.
- Methods for maintenance in cell culture if applicable: The strain cultures are stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen. From the thawed ampoules of the strains 0.5 mL bacterial suspension were transferred to 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 µL ampicillin (25 µg/mL) was added to the strains TA 98 and TA 100. The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The nutrient medium contains per litre: 8 g Merck Nutrient Broth and 5 g NaCl.
The overlay agar contains per litre:
For Salmonella strains:
6.0 g Merck agar
6 g NaCl
10.5 mg L-histidine·HCl·H2O
12.2 mg biotin
For E. coli strain:
6.0 g Merck agar
6 g NaCl
2.5 mg tryptophan
Sterilisation was performed at 121 °C in an autoclave.

- Properly maintained: Yes.
- Regular checking of the properties of the S. typhimurium and E. coli trains regarding the membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed at the testing facility.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 mix and hamster liver S9 mix.

>> Rat Liver S9 (Preparation by RCC Cytotest Cell Research)
Phenobarbital/ß-napthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar Han/bm rats, weight approx. 220 – 320 g induced by applications of 80 mg/kg b.w. phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
The protein concentration was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9-preparation was 39.9 mg/mL.
 
>> Hamster Liver S9 (Preparation by RCC Cytotest Cell Research)
The S9 liver microsoma fraction was prepared from the liver of 7 – 8 weeks old male Syrian golden hamsters.
After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9000 g for 25 minutes at 4 °C. Aliquots of the supernatant were frozen and stored in ampoules at -80°C. Small numbers of ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
The protein concentration was determined using an analysis kit of Bio-Rad Laboratories. The protein concentration in the S9-preparation was 34.7 mg/mL.
 
>> Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 15 % v/v. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
In 100 mM sodium-ortho-phosphate buffer, pH 7.4.
During the experiment the S9 was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
 
>> Hamster S9 mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30 % v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
20 mM glucose-6-phosphate
2.8 units /mL glucose-6-phosphate dehydrogenase
4 mM NADP
2 mM NADH
2 mM FMN
In 100 mM sodium-ortho-phosphate buffer, pH 7.4.
During the experiment the S9 was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. and Prival and Mitchell.

Test concentrations with justification for top dose:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

In the pre-experiment the concentration range of the test material was 3 – 5 000 µg/plate. The pre-experiment is reported as experiment I since the criteria were met and 5 000 µg/plate was chosen as maximal concentration.
The concentration range included two logarithmic decades.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Remarks:

Concurrent untreated

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-pheylene-diamine in DMSO without metabolic activation 50 µg/plate in TA 1537 and 10 µg/plate with TA 98.

Remarks:

The stability of the positive control substances in solution is unknown but a mutagenic response in the expected range willl be sufficient evidence of biological stability.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation (experiment I) and preincubation (experiment II)

DURATION
- Preincubation period: 30 minutes in experiment II
- Selection time (if incubation with a selection agent): At least 48 h at 37 °C in the dark.

The following materials were mixed in a test tube and poured onto the selective agar plates (first experiment):
100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control);
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation);
100 µL bacterial suspension (cf. test system, pre-culture of the strains);
2000 µL overlay agar.
According to the pre-incubation method (second experiment) 100 µL test solution, 500 µL S9 mix/S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 30 °C for 30 minutes. After pre-incubation 2 mL overlay agar was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 h at 37 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
Pre-Experiment for Toxicity
To evaluate the toxicity of the test material a pre-experiment was performed with all strains. Eight concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as the main experiment.
Toxicity of the test material resulted in a reduction in the number of spontaneous revertants or a clearing of the bacterial lawn.
The pre-experiment is reported as the main experiment I if the following criteria are met:
Evaluable plates (> 0 colonies) at five concentrations or more.

OTHER EXAMINATIONS
The colonies were counted using the Petri Viewer Mk2 (Perspective Instruments Ltd.) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer to print out the individual and mean values from the plates of each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation and the intense colour of the test material the revertant colonies were partly counted manually.

Evaluation criteria:

Acceptability of the Assay
The S. typhimurium and E. coli reverse mutation assay is considered acceptable if it meets the following criteria:
- Regular background growth in the negative and solvent control.
- The spontaneous reversion rates in the negative and solvent control are in the range of historical data.
- The positive control substances should produce a significant increase in mutant colony frequencies.

Evaluation of results
A test material is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, when the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
- No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test material precipitated in the overlay agar at 2500 and 5000 µg/plate in Experiment I, with and without S9 mix, and in Experiment II with S9 mix. The undissolved particles had no influence on the data recording.

HISTORICAL CONTROL DATA
The laboratory’s historical control range was slightly exceeded in the untreated control of strain WP2 uvrA with metabolic activation in Experiment II. This deviation is judged to be based on biologically irrelevant fluctuations in the number of colonies and has no impact on the outcome of the study.

Precipitation

The test material precipitated in the overlay agar at the following concentrations (µg/plate)

Strain	Experiment I		Experiment II
	Without S9 mix	With S9 mix	Without S9 mix	With S9 mix
TA 1535	2500, 5000	2500, 5000	-	2500, 5000
TA 1537	2500, 5000	2500, 5000	-	2500, 5000
TA 98	2500, 5000	2500, 5000	-	2500, 5000
TA 100	2500, 5000	2500, 5000	-	2500, 5000
WP2uvrA	2500, 5000	2500, 5000	-	2500, 5000

The undissolved particles had no influence on the data recording.

Conclusions:

Under the conditions of the study the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic.

Executive summary:

The mutagenic potential of the test material was assessed according to OECD Test Guideline 471 and Japanese Guidelines and in compliance with GLP using an Ames test.

This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including controls, was tested in triplicate. The test material was tested at the following concentrations:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Under the conditions of the study, the plates incubated with the test material showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. 

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence not absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutations were used as positive controls and showed a distinct increase of induced revertant colonies.

It was therefore concluded that the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames: Sokolowski (2006)

The mutagenic potential of the test material was assessed according to OECD Test Guideline 471 and Japanese Guidelines and in compliance with GLP using an Ames test. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including controls, was tested in triplicate. The test material was tested at the following concentrations:

Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate.

Under the conditions of the study, the plates incubated with the test material showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. 

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test material at any dose level, neither in the presence not absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutations were used as positive controls and showed a distinct increase of induced revertant colonies.

It was therefore concluded that the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.