1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102 both without and with S9-mix.

Chromosome aberration:

1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in Chinese hamster ovary cells and mouse lymphoma both without and with S9-mix.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

WoE report is based on two in-vitro gene toxicity studies on rats
1. The experiments were performed to assess the potential of the test item to induce gene mutations by means of two independent Salmonella typhimurium reverse mutation assays.
Experiment I was performed as a plate incorporation assay. Since a negative result was obtained in this experiment, experiment II was performed as a pre-incubation assay.
2. In virto gene toxicity study of test chemical in Salmonella typhimurium

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

1. The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includesa deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100, and TA 102 the Rfactor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrB--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation (ochre mutation in the hisG gene ) and a tetracycline resistance gene (5).In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA 1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA 98 his D 3052; rfa-; uvrB-;R-factor " "
TA 1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA 102 his G 428; rfa-; uvrB+;R-factor " "
TA 100 his G 46; rfa-; uvrB-;R-factor " "
Regular checking of the properties of the strains regarding the membrane permeability,ampicillin- and tetracycline resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al.and D.Maron and B. Ames.In this way it was ensured
that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537 TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Species / strain / cell type:

S. typhimurium TA 1535

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 98

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 102

Remarks:

1.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

2.

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not specified

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal activation

Test concentrations with justification for top dose:

1. In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as part of experiment I. Since the criteria mentioned above were met 5000 μg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

2.experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix
experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used:
With metabolic activation
Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 - DMSO
Without metabolic activation
Strains: TA 1535, TA 100 - water deionised
Strains: TA 1537, TA 98 - DMSO
Strain: TA 102 - water deionised
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

2.DMSO

Untreated negative controls:

yes

Remarks:

1.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 10 μg/plate

Positive control substance:

sodium azide

Remarks:

For Strains: TA 1535, TA 100 without metabolic activation

Untreated negative controls:

yes

Remarks:

1.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 10 μg/plate in TA 98, 50 μg/plate in TA 1537

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

For Strains: TA 1537, TA 98 without metabolic activation

Untreated negative controls:

yes

Remarks:

1.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 4.0 μL/plate

Positive control substance:

methylmethanesulfonate

Remarks:

For Strain: TA 102 without metabolic activation

Untreated negative controls:

yes

Remarks:

1.

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

Concentration: 2.5 μg/plate (10.0 μg/plate in TA 102)

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

For Strains: TA 1535, TA 1537, TA 98, TA 100, TA 102 with metabolic activation

Untreated negative controls:

yes

Remarks:

2.

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: in accordance with the OECD guideline

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as mpregnation on paper disk
Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.Precultures
From the thawed ampoules of the strains 0.5 mL bacterial suspension was transferred into 250 mL Erlen meyer flasks containing 20 mL nutrient medium. A solution of 20 μL ampicillin (25 μg/mL) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 μL tetracycline (2 μg/mL) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.
Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.
Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121° C in an autoclave.
Experimental Performance
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 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 Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 μL Overlay agar
In the pre-incubation assay 100 μL test solution, 500 μL S9 mix / S9 mix substitution buffer and 100μL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-in
cubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark .

2.Details on test system and conditions
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk
Experiment I was performed according to the direct plate incorporation test, experiment II with the preincubation method.
DURATION
Preincubation period: 60 minutes
Exposure duration: 48 hours incubation with and without S9 mix
NUMBER OF REPLICATIONS: triplicates in 2 individual experiments
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
Determination of polyploidy: No data
Determination of endoreplication: No data
Other:
experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix
Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because
relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I.

Evaluation criteria:

1. The Salmonella typhimurium 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 our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
Evaluation of Results
A test item 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 TA 102) or thrice (strains TA 1535 and TA1537) 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 reprod uced 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,
whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

1.According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

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

Remarks:

1.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Remarks:

2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102 both without and with S9-mix.

Executive summary:

Study 1

This study was performed to investigate the potential of test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

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

Reduced background growth was observed with and without S9 mix in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without S9 mix in all strains used (cf. tables of results).

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor 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 and showed a distinct increase of induced revertant colonies. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test chemical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Study 2

Test chemcail was investigated for the induction of gene mutations in strains of Salmonella typhimurium(Ames test).

Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 were used for the AMES assay. DMSO was used as a vehicle. Concentration: experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix

Treatment: experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix. Liver S9-fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I. Experiment I was performed according to the direct plate incorporation test, experiment II with the pre-incubation method. Negative and positive controls were in accordance with the OECD guideline.

In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Under the experimental conditions used, test chemical was not mutagenic in this gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation.

Test chemical induced a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA1537, and TA98 in the presence of S9 metabolic activation system. No mutagenic potential was observed for the tester strain TA1537 in the presence and absence of S9 metabolic activation system. Based on these observations, test chemical is likely to be mutagenic.

Based on the data available from the read across, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data of read across substances

Justification for type of information:

Weight of evidence approach based on structurally similar chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2.

Principles of method if other than guideline:

WoE report is based on two in-vitro gene toxicity studies on rats
1. The genetic toxicity of test chemical was assessed by testing the ability of the chemical to induce chromosome aberration in Chinese hamster ovary cells.
2. In -vitro mammalian cell gene mutation test of test chemical

GLP compliance:

not specified

Type of assay:

other: 1. chromosome aberration test, 2. mammalian cell gene mutation assay

Target gene:

2. Mammalian genetox assay using the tk locus as reporter gene.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

mammalian cell line 1.

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

mammalian cell line 2.

Details on mammalian cell type (if applicable):

not specified

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

1. 0,30, 50, 100 μg/ml
2. experiment I: 26.6, 53.1, 106.3, 212.5 and 318.8 μg/ml without and with S9-mix
experiment II: 53.1, 106.3, 212.5, 318.8 and 425.0 μg/ml without S9-mix

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO
2. Deionized water

Untreated negative controls:

not specified

Remarks:

1.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: in accordance with the OECD guideline

Details on test system and experimental conditions:

1. Details on test system and conditions
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data available
- Exposure duration:
Without metabolic activation- 15 hour
With metabolic activation- 2 hour
- Expression time (cells in growth medium): Without metabolic activation- 15 hour
With metabolic activation- 13 hour
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays):stained with Giemsa
OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other:

2. METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk experiment I: 4 h both without and with S9-mix; expression period 72 h, selection growth 10-15 days experiment II: 24 h without S9-mix; expression period 48 h, selection growth 10-15 days.
NUMBER OF REPLICATIONS:
duplicate cultures in 2 independent experiments
OTHER: The test concentrations were based on the results of a pre-test with treatments up to 1700 μg/ ml both in the presence and absence of S9-mix measuring suspension growth relative to the concurrent vehicle control cell cultures. In the main test, cells were treated for 4 h (experiment I: both without and with S9-mix) or 24 h (experiment II: without S9-mix only), followed by an expression period of 72 h (experiment I) or 48 h (experiment II) to fix the DNA damage into a stable tk mutation and a selection growth 10-15 days. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed.
If the increase in mutant frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. Toxicity was measured in the main experiments as percentage relative total growth and relative suspension growth of the treated cultures.

Evaluation criteria:

1. Classes of aberrations included simple (breaks and terminal deletions), complex (rearrangements and translocations), and other (pulverized cells, despiralized chromosomes, and cells containing 10 or more aberrations). were observed .

Statistics:

Not applicable.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

1.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

mammalian cell line 2.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

2. TEST-SPECIFIC CONFOUNDING FACTORS
No relevant increase of the osmolarity and pH-value was observed at the maximum concentration.
RANGE-FINDING/SCREENING STUDIES:
In the pre-test after 4 h treatment, precipitation was observed at 425 μg/ml both in the absence and presence of S9-mix and at 212.5 μg/ml after 24 h treatment in the absence of S9-mix. No relevant increase of the osmolarity and pH-value was observed at the maximum concentration.

Remarks on result:

other: No mutagenic effect were observed

Conclusions:

1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in Chinese hamster ovary cells and mouse lymphoma both without and with S9-mix.

Executive summary:

Study 1

The genetic toxicity of test chemical was assessed by testing the ability of the chemical to induce chromosome aberration in Chinese hamster ovary cells. In the Abs test without S9, cells were incubated in McCoy's 5A medium with 30, 50, 100 µg/ml concentration of test chemical for 10-h Colcemid was added and incubation continued for 2 to 3 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with 30, 50, 100 µg/ml concentration of test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. No induction of chromosome aberration was observed in Chinese hamster ovary cell in presence and absence of metabolic activation.

Study 2:

Test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation.

Mouse lymphoma cell line L5178Y was used for the assay. Deionized water was used as vehicle.  

Concentrations: experiment I: 26.6, 53.1, 106.3, 212.5 and 318.8 μg/ml without and with S9-mix experiment II: 53.1, 106.3, 212.5, 318.8 and 425.0 μg/ml without S9-mix

Treatment: experiment I: 4 h both without and with S9-mix; expression period 72 h, selection growth 10-15 days experiment II: 24 h without S9-mix; expression period 48 h, selection growth 10-15 days.

Liver S9-fraction from phenobarbital/β- naphthoflavone-induced rats was used as exogenous metabolic activation system. test concentrations were based on the results of a pre-test with treatments up to 1700 μg/ml both in the presence and absence of S9-mix measuring suspension growth relative to the concurrent vehicle control cell cultures. In the main test, cells were treated for 4 h (experiment I: both without and with S9-mix) or 24 h (experiment II: without S9-mix only), followed by an expression period of 72 h (experiment I) or 48 h (experiment II) to fix the DNA damage into a stable tk mutation and a selection growth 10-15 days. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed. If the increase in mutant frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. Toxicity was measured in the main experiments as percentage relative total growth and relative suspension growth of the treated cultures. Negative and positive controls were in accordance with the OECD guideline. In the pre-test after 4 h treatment, precipitation was observed at 425 μg/ml in the absence and presence of S9-mix and at 212.5 μg/ml after 24 h treatment in the absence of S9-mix. No relevant increase of the osmolarity and pH-value was observed at the maximum concentration. In both experiments in the absence and presence of S9-mix the appropriate level of toxicity (about 10-20% survival after the highest dose) was reached; only in experiment II in one culture treated with the highest concentration of the test chemical, the appropriate level of toxicity was not reached. Both in the absence and presence of metabolic activation, a biologically relevant increase in the mutant frequency due to exposure to the test chemical was not reported. Under the experimental conditions used, test chemical was not mutagenic in this mouse lymphoma assay using thetklocus as reporter gene.

Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene toxicity: in-vitro

Data available for the read across chemicals was reviewed to determine the in- vitro gene toxicity of 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates (CAS no. 84281-74-3). The studies are as mentioned below:

Ames test:

Study 1

This study was performed to investigate the potential of test chemical to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98,TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment I: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

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

Reduced background growth was observed with and without S9 mix in strains TA 98 and TA 100 in experiment I. In experiment II, reduced background growth was observed with and without S9 mix in all strains used (cf. tables of results).

Toxic effects, evident as a reduction in the number of revertants, were observed at higher concentrations with and without metabolic activation in nearly all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test chemical at any dose level, neither in the presence nor 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 and showed a distinct increase of induced revertant colonies. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test chemical is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Study 2

Test chemcail was investigated for the induction of gene mutations in strains of Salmonella typhimurium(Ames test).

Salmonella typhimuriumTA98, TA100, TA102, TA1535 and TA1537 were used for the AMES assay. DMSO was used as a vehicle. Concentration: experiment I: 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate without and with S9-mix

Treatment: experiment I: direct plate incorporation with 48 h incubation without and with S9-mix experiment II: pre-incubation method with 60 minutes pre-incubation and at least 48 h incubation without and with S9-mix. Liver S9-fraction from phenobarbital/β-naphthoflavone-induced rats was used as exogenous metabolic activation system. Test concentrations were based on the results of a pre-experiment with strains TA98 and TA100 for toxicity and mutation induction both without and with S9-mix. Toxicity was evaluated for 8 concentrations up to the prescribed maximum concentration of 5000 μg/plate on the basis of a reduction in the number of revertant colonies and/or clearing of the bacterial background lawn. Because relevant toxic effects were not observed in any of the strains at the maximal concentration, 5000 μg/plate was used as the top concentration. Since in this pre-experiment evaluable plates were obtained for five concentrations or more in the strains used, the pre-experiment is reported in experiment I. Experiment I was performed according to the direct plate incorporation test, experiment II with the pre-incubation method. Negative and positive controls were in accordance with the OECD guideline.

In the main tests toxic effects evident as clearing of the bacterial background lawn were observed in experiment I in TA98 and TA100 and in experiment II in all strains predominantly at higher concentrations. Toxic effects evident as a reduction in the number of revertants were observed at higher concentrations without and with metabolic activation in nearly all strains tested. A biologically relevant increase in revertant colonies was not observed in any of the strains tested at any dose level in the absence or presence of S9-mix in both experiments. Under the experimental conditions used, test chemical was not mutagenic in this gene mutation tests in bacteria both in the absence and the presence of S9 metabolic activation.

Test chemical induced a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA1537, and TA98 in the presence of S9 metabolic activation system. No mutagenic potential was observed for the tester strain TA1537 in the presence and absence of S9 metabolic activation system. Based on these observations, test chemical is likely to be mutagenic.

Based on the data available from the read across, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation. 

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of diazotized m-phenylenediamine, acetates (CAS no. 84281-74-3) when administered to Chinese Hamster Ovary (CHO) cells.

Study 1

The genetic toxicity of test chemical was assessed by testing the ability of the chemical to induce chromosome aberration in Chinese hamster ovary cells. In the Abs test without S9, cells were incubated in McCoy's 5A medium with 30, 50, 100 µg/ml concentration of test chemical for 10-h Colcemid was added and incubation continued for 2 to 3 hours. The cells were then harvested by mitotic shake-off, fixed, and stained with Giemsa. For the Abs test with S9, cells were treated with 30, 50, 100 µg/ml concentration of test chemical and S9 for 2 hours, after which the treatment medium was removed and the cells incubated for 11 hours in fresh medium, with Colcemid present for the final 2 hours. No induction of chromosome aberration was observed in Chinese hamster ovary cell in presence and absence of metabolic activation.

Study 2:

Test chemical was assayed for gene mutations at the tk locus of mouse lymphoma cells both in the absence and presence of S9 metabolic activation.

Mouse lymphoma cell line L5178Y was used for the assay. Deionized water was used as vehicle.  

Concentrations: experiment I: 26.6, 53.1, 106.3, 212.5 and 318.8 μg/ml without and with S9-mix experiment II: 53.1, 106.3, 212.5, 318.8 and 425.0 μg/ml without S9-mix

Treatment: experiment I: 4 h both without and with S9-mix; expression period 72 h, selection growth 10-15 days experiment II: 24 h without S9-mix; expression period 48 h, selection growth 10-15 days.

Liver S9-fraction from phenobarbital/β- naphthoflavone-induced rats was used as exogenous metabolic activation system. test concentrations were based on the results of a pre-test with treatments up to 1700 μg/ml both in the presence and absence of S9-mix measuring suspension growth relative to the concurrent vehicle control cell cultures. In the main test, cells were treated for 4 h (experiment I: both without and with S9-mix) or 24 h (experiment II: without S9-mix only), followed by an expression period of 72 h (experiment I) or 48 h (experiment II) to fix the DNA damage into a stable tk mutation and a selection growth 10-15 days. To discriminate between large (indicative for mutagenic effects) and small colonies (indicative for a clastogenic effect) colony sizing was performed. If the increase in mutant frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. Toxicity was measured in the main experiments as percentage relative total growth and relative suspension growth of the treated cultures. Negative and positive controls were in accordance with the OECD guideline. In the pre-test after 4 h treatment, precipitation was observed at 425 μg/ml in the absence and presence of S9-mix and at 212.5 μg/ml after 24 h treatment in the absence of S9-mix. No relevant increase of the osmolarity and pH-value was observed at the maximum concentration. In both experiments in the absence and presence of S9-mix the appropriate level of toxicity (about 10-20% survival after the highest dose) was reached; only in experiment II in one culture treated with the highest concentration of the test chemical, the appropriate level of toxicity was not reached. Both in the absence and presence of metabolic activation, a biologically relevant increase in the mutant frequency due to exposure to the test chemical was not reported. Under the experimental conditions used, test chemical was not mutagenic in this mouse lymphoma assay using thetklocus as reporter gene.

Based on the data available from the read across, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available from the read across, 1,3-Benzenediamine, coupled with diazotized m-phenylenediamine, acetates does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.