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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
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

Test material

impurity 1
Chemical structure
Reference substance name:
Melamine
EC Number:
203-615-4
EC Name:
Melamine
Cas Number:
108-78-1
Molecular formula:
C3H6N6
Constituent 1
Chemical structure
Reference substance name:
6-phenyl-1,3,5-triazine-2,4-diyldiamine
EC Number:
202-095-6
EC Name:
6-phenyl-1,3,5-triazine-2,4-diyldiamine
Cas Number:
91-76-9
Molecular formula:
C9H9N5
IUPAC Name:
6-phenyl-1,3,5-triazine-2,4-diamine

Method

Target gene:
his-
Species / strain
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
Controls
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

Results and discussion

Test resultsopen allclose all
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

Any other information on results incl. tables
































































































































































































































































































































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

Applicant's summary and conclusion

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.