6-phenyl-1,3,5-triazine-2,4-diyldiamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

Experimental Starting date: 28. July 2021
Experimental Completion date: 13. August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, EU-Method B.13/14 adopted 30. May 2008 “Mutagenicity –Reverse mutation test using bacteria”

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for the Testing of Chemicals Part 471, adopted 26. Jun. 2020
“Bacterial Reverse Mutation Test“

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix [(+S9) and (-S9)] produced from the livers of male Sprague-Dawley rats which were treated with Phenobarbital/5,6-Benzoflavone

Test concentrations with justification for top dose:

Experiment 1: 5000, 1500, 500, 150, 50 µg/plate
Experiment 2: 5000, 2500, 1250, 625, 313, 156 µg/plate
Experiment 3: 5000, 2500, 1250, 625, 313, 156 µg/plate

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

DMSO was chosen as solvent, because the Benzoguanamine was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of sponta-neous revertants

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other:

Rationale for test conditions:

according to guideline

Evaluation criteria:

Five different analyzable and non-toxic concentrations were used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination were calculat-ed as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given.
A result is considered as clearly positive if all following criteria are fulfilled:
• A concentration-related increase in revertants
• a clear biological relevant increase in at least one concentration compared to the concurrent solvent control
• at least one concentration with an increase above the distribution of historical sol-vent control data (mean ± 3 SD).

A biologically relevant increase is described as follows:
• if in the bacteria strains TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
• if in the bacteria strains TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

A test result is considered as clearly negative, if it does not meet the criteria above.

Statistics:

no statistic evaluation performed

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	14	17	98	96	183	179	7	6	3	4
	sd	1.0	0.6	4.0	4.0	8.3	8.3	1.0	0.6	0.6	0.6
DMSO	Mean	14	16	95	94	181	179	7	8	3	4
	sd	1.0	1.7	2.3	2.0	12.2	11.5	0.6	1.0	0.6	1.0
Positive Controls*	Mean	731	83	451	1099	577	565	209	143	92	69
	sd	28.1	8.3	16.7	20.1	18.9	18.9	10.1	18.0	8.0	10.1
	f(I)	52.21	5.19	4.60	11.69	3.15	3.16	29.86	17.88	30.67	17.25
5000 µg/plate	Mean	13	13	85	79	160	161	6	7	3	4
	sd	1.0	0.0	2.3	4.6	12.0	6.1	0.6	1.0	0.0	0.6
	f(I)	0.93	0.81	0.89	0.84	0.88	0.90	0.86	0.88	1.00	1.00
1500 µg/plate	Mean	14	17	80	81	177	175	6	6	4	4
	sd	0.6	0.6	4.0	6.1	10.1	12.2	0.6	0.6	0.0	0.6
	f(I)	1.00	1.06	0.84	0.86	0.98	0.98	0.86	0.75	1.33	1.00
500 µg/plate	Mean	14	15	79	83	173	181	8	7	4	4
	sd	2.0	2.1	6.1	2.3	12.2	8.3	0.6	0.6	1.0	0.6
	f(I)	1.00	0.94	0.83	0.88	0.96	1.01	1.14	0.88	1.33	1.00
150 µg/plate	Mean	17	14	80	79	169	173	7	8	3	4
	sd	1.7	0.6	4.0	6.1	6.1	6.1	0.6	1.7	0.6	0.6
	f(I)	1.21	0.88	0.84	0.84	0.93	0.97	1.00	1.00	1.00	1.00
50 µg/plate	Mean	14	16	79	75	168	176	8	8	3	3
	sd	1.0	1.5	9.2	2.3	8.0	8.0	0.0	0.6	0.0	0.6
	f(I)	1.00	1.00	0.83	0.80	0.93	0.98	1.14	1.00	1.00	0.75

sd = standard deviation ±

* Different positive controls were used

f(I) = increase factor

Conclusions:

Based on the results of this study it is concluded that 6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine) is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

The test was performed in three valid experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation and -S9 standing for absence of metabolic activation.

Experiment 1: In the first experiment, the test item (dissolved in Dimethyl sulfoxide, DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation.The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2: Based on the results of exp. 1, the test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-incubation method.The test item showed no precipitates on the plates at any of the concentrations and no signs of cytotoxicity could be observed in the presence and the absence of metabolic activation.The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Due to a technical error (evaluation not possible), the exp. 2 was repeated for the strain TA98 with and without metabolic activation (exp. 2b).

Experiment 2b: The test item was tested up to concentrations of 5000 µg/plate in the presence and absence of S9 mix in the strain TA98 using the pre-incubation method.The test item showed no precipitates on the plates at any of the concentrations and cytotoxicity could be observed in absence of metabolic activation in TA98 (-S9). The results of this experiment showed that none of the tested concentrations induced a relevant or concentration-related increase in the number of revertants in the strain TA98, in the presence and the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Benzoguanamine was tested using the Ames test according to OECD TG 471 and GLP and showed no mutagenic activity neither with nor without exogenous metabolic activation up to 5000 µg/plate. In six studies no evidence of mutagenic activity of Benzoguanamine with and without mutagenic activation was observed (Anonym 1988, Clare 1988, Nakajima 1999, Himmelsbach_2021).

In addition, a study was preformed to investigate the potential of Benzoguanamine to induced gene mutation at the mouse lymphoma cells line L 5178 TK+/- according to OECD TG 476 and GLP in concentration up to 2500 µg/mL in the presence and absence of S9 -mix. Cytotoxicity was observed in the test with metabolic activation above the solubility limit. No Cytotoxicity was found in the test with metabolic activation within the solubility limit and in the test without metabolic activation (Jenkinson 1993).

According to OECD TG 473 and GLP two test were preformed.

In the first test Benzoguanamine was tested for chromosomal abberation with human lymphocytes cells in concentrations up to 2500 µg/mL with and without metabolic activation system. There was no evidence for cytotoxicity with metabolic activation system within and above the solubility limit. Cytotoxicity was detected without metabolic activation system within and above the solubility limit (Pateman 1993).

In the second test Benzoguanamine was tested for chromosomal abberation in Chinese Hamster Lung (CHL) cells with and without metabolic activation system. Cytotoxicity was observed without metabolic activation system. Findings for cytotoxicity with metabolic activation system was ambiguous (Nakajima 1999).

The cytogenetic effect observed in in vitro assays however, could not be reproduced in the micronucleus tests in vivo (Honarvar 2000).

Short description of key information:
Benzoguanamine revealed no mutagenic activity in the Ames test according to OECD TG 471 and GLP.
Cytogenic effects were observed when tested according to OECD TG 476 and 473. The cytogenetic effects, observed in in-vitro assays, could not be reproduced in the in vivo micronucleus tests. Based on these results, it could be concluded that Benzoguanamine is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

For genotoxicity Benzoguanamine is not classified in accordance to Directive 67/548/EEC, respectively Regulation (EC) No 1272/2008.