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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) is non mutagenic to the bacterial tester strain E. coli 343/113.

Link to relevant study records
Reference
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer-reviewed journal.
Qualifier:
no guideline available
Principles of method if other than guideline:
In –vitro forward and reverse bacterial gene mutation assay for 2-chloro-p-phenylenediamine was studied in E. coli.

GLP compliance:
not specified
Type of assay:
bacterial gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4 –diamino-2-chlorbenzol
- Molecular formula: C6-H7-Cl-N2
- Molecular weight: 142.588 g/mol
- Substance type: Organic
- Physical state: Solid
- Purity: 99%
- Impurities (identity and concentrations): 1 %
Target gene:
343/113
Species / strain / cell type:
E. coli, other: E. coli 343/113
Details on mammalian cell type (if applicable):
No data available
Additional strain / cell type characteristics:
not specified
Metabolic activation:
not specified
Test concentrations with justification for top dose:
1, 10 and 100 μg/ml
Vehicle / solvent:
Vehicle
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No data available
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
Details on test system and conditions
METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Preincubation period: No data available
- Exposure duration: 2 hours
- Expression time (cells in growth medium): 20 hours for standard medium, of 40 hours for gal+ mutants, and of 72 hours arg+ , nad+ and MTR mutants.
- Selection time (if incubation with a selection agent): 3 hours
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: Duplicate cultures per “mutant” type in one experiment

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data available

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: Both forward and reverse mutations in different genes: reverse mutations of arg- and nad- to prototrophy, forward mutations of 5-methyl-DL-tryptohan sensitivity (MTS) to MT resistance and forward (and reverse) mutations from Rs 18 to gal+.
Evaluation criteria:
A biologically relevant increase in the number of revertants
Statistics:
No data available
Species / strain:
E. coli, other: E. coli 343/113
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data available
- Effects of osmolality: No data available
- Evaporation from medium: No data available
- Water solubility: No data available
- Precipitation: No data available
- Other confounding effects: No data available

RANGE-FINDING/SCREENING STUDIES: Test concentrations were based on the basis of survival in a toxicity test with 3 h incubation with 1, 4–diamino-2-chlorbenzol in the dark.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: no mutagenic potential
Conclusions:
The substance 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) is non mutagenic to the bacterial tester strain E. coli 343/113.

Executive summary:

In a in –vitro forward and reverse bacterial gene mutation assay, E. coli343/113 was treated with 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) in concentration of 1, 10 and 100 μg/ml. No effect were observed on survival of E. coli343/113 during 2 hours incubation and an expression period of 20 hours for standard medium, of 40 hours for gal+mutants, and of 72 hours arg+, nad+and MTR mutants for forward mutations of 5-methyl-DL-tryptohan sensitivity (MTS) to MT resistance and forward (and reverse) mutations from Rs18to gal+.

 

The substance 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) is non mutagenic to the bacterial tester strain E. coli 343/113.

 

According to the CLP classification, the test material does not classify as a gene mutant in vitro.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) failed to induce an increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow cells and hence is negative for gene mutation in vivo.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
Type of genotoxicity: Gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from SCCS report
Qualifier:
no guideline available
Principles of method if other than guideline:
In vivo Bone marrow micronucleus test for 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) was performed in rats.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 1,4-diamino-2-chlorobenzene
- Molecular formula: C6-H7-Cl-N2
- Molecular weight: 142.588 g/mole
- Substance type: Organic
- Physical state: Solid
- Purity: 99%
- Impurities (identity and concentrations): 1 %
Species:
rat
Strain:
other: CFY SPF
Sex:
male/female
Details on test animals and environmental conditions:
No data available
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% gum tragacanth
- Justification for choice of solvent/vehicle: No data available
- Concentration of test material in vehicle: 0 and 900 mg/kg bw/day
- Amount of vehicle (if gavage or dermal): No data available
- Type and concentration of dispersant aid (if powder): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available
Details on exposure:
No data available
Duration of treatment / exposure:
30 hours
Frequency of treatment:
Twice in 24h interval
Post exposure period:
6 hours
Remarks:
Doses / Concentrations:
0 and 900 mg/kg bw/day
Basis:
no data
No. of animals per sex per dose:
5/sex/dose
Total: 20
0 mg/kg bw/day: 5 male, 5 female
900 mg/kg bw/day: 5 male, 5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
No data available
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Test doses were based on the results of a preliminary toxicity study with doses between 800 and 900 mg/kg bw/day on a group of 5 male and 5 female rats recording clinical signs and mortality for a period of 30 h performed under identical conditions.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): No data available

DETAILS OF SLIDE PREPARATION: No data available

METHOD OF ANALYSIS: No data available

OTHER: No data available
Evaluation criteria:
Increase in the number of bone marrow cells with micronuclei
Statistics:
No data available
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
not specified
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: between 800- 900 mg/kg bw/day
- Solubility: No data available
- Clinical signs of toxicity in test animals: Orange pigmented urine was observed.
- Evidence of cytotoxicity in tissue analyzed: No data available
- Rationale for exposure: No data available
- Harvest times: 30 hours
- High dose with and without activation: 900 mg/kg bw/day
- Other: No data available

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No data available
- Induction of micronuclei (for Micronucleus assay): Biologically relevant increases in the number of polychromatic erythrocytes with micronuclei compared to the concurrent vehicle controls were not found in either males or females.
- Ratio of PCE/NCE (for Micronucleus assay): No data available
- Appropriateness of dose levels and route: No data available
- Statistical evaluation: No data available
Conclusions:
The substance 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) failed to induce an increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow cells and hence is negative for gene mutation in vivo.
Executive summary:

In a in -vivo gene toxicity test, CFY SPF male and female rats were treated with 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) in the concentration of 0 and 900 mg/kg bw/day. Bone marrow cells were collected 6 h after the last treatment. For each rat the percentage of polychromatic erythrocytes with a micronucleus was counted in 2000 polychromatic erythrocytes. Negative and positive controls were included.

 

2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) failed to induce an increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow cells and hence is negative for gene mutation in vivo.

 

According to the CLP classification, the test material does not classify as a gene mutant in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The potential genotoxicity of 2-chloro-p-phenylenediamine (CAS No. 615-66-7) was evaluated using in vitro and in vivo assays. Various peer reviewed publications and prediction model based estimation for target substance and read across substances have been reviewed and summarized to determine the mutagenic potential of 2-chloro-p-phenylenediamine:

In Vitro

2-chloro-p-phenylenediamine was investigated for the induction of gene mutations in E. coli strain 343/113 according to Mohn et al. (Mutation Research 25, 187-196, 1974). In a in –vitro forward and reverse bacterial gene mutation assay, E. coli343/113 was treated with 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) in concentration of 1, 10 and 100 μg/ml. No effect were observed on survival of E. coli343/113 during 2 hours incubation and an expression period of 20 hours for standard medium, of 40 hours for gal+mutants, and of 72 hours arg+, nad+and MTR mutants for forward mutations of 5-methyl-DL-tryptohan sensitivity (MTS) to MT resistance and forward (and reverse) mutations from Rs18to gal+. The substance 2-chloro-p-phenylenediamine (1,4–diamino-2-chlorbenzol) is non mutagenic to the bacterial tester strain E. coli 343/113.  

 

Also, the gene mutation study of 2-chloro-p-phenylenediamine (CAS No. 615-66-7) was predicted using OECD QSAR toolbox version 3.3 (2017) with respect to the descriptor log Kow and considering the ten closest read across substances. This is specifically designed to assess the ability of a chemical to cause point mutations in the DNA of the bacterium Salmonella typhimurium strains. The prediction assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the presence of S9 mix. The substance 2-chloro-p-phenylenediamine was predicted to be non mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence of S9 metabolic activation system.

Similarly, the gene mutation study of 2-chloro-p-phenylenediamine (CAS No. 615-66-7) was predicted using OECD QSAR toolbox version 3.3 (2017) with respect to the descriptor log Kow and considering the ten closest read across substances. The prediction assumed the use of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the absence of of S9 mix. The substance 2-chloro-p-phenylenediamine was predicted to be non mutagenic in S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the absence of S9 metabolic activation system.

 

In addition to these studies, Zeiger,E et al (Environ. Mutagen. Vol. 9 (Suppl 9) (1987) 1-109) conducted gene mutation study for read across substance 3-chloroaniline. In an Ames test , 3-chloroaniline, in dimethyl sulfoxide from doses 33 - 2000 µg/plate was not mutagenic in Salmonella typhimurium strains TA100 , TA 1535, TA1537 , TA 97 and TA 98 with and without addition of S9 liver microsome fractions from Aroclor induced rats.The same has been obtained for hamsters as well.

 

Moreover,Loveday et al (Environ. Mol. Mutagen. 16(9):272-303,1990) conducted mammalian chromosome aberration test . In mammalian chromosome aberration test , 4-chlorobenzene-1,3-diamine, in dimethyl sulfoxide from doses 148; 494; 14800 µg/ml was not mutagenic in CHO-LB CELLS with addition of S9 liver microsome fractions from Aroclor induced rats.

Based on the key study and its supporting data summarized,the test material does not classify as a gene mutant in vitro.

In Vivo

2-chloro-p-phenylenediamine has been investigated for induction of micronuclei in bone marrow cells of rats. The study was published in a Scientific Committee on Consumer Safety report (OPINION ON 2-Chloro-p-phenylenediamine COLIPA n° A8). In this in -vivo gene toxicity test, CFY SPF male and female rats were treated with 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) in the concentration of 0 and 900 mg/kg bw/day. Bone marrow cells were collected 6 h after the last treatment. For each rat the percentage of polychromatic erythrocytes with a micronucleus was counted in 2000 polychromatic erythrocytes. Negative and positive controls were included. 2-chloro-p-phenylenediamine (1,4-diamino-2-chlorobenzene) failed to induce an increase in the number of polychromatic erythrocytes with micronuclei in the bone marrow cells and hence is negative for gene mutation in vivo.  

 

Moreover, gene toxicity in vivo study was conducted by W. Suter et al. (Environmental and Molecular Mutagenesis 28:354-362 (1996)) on male and female Big BlueTMmice. The test compound 4-chloro-o-phenylenediamine (4-C-o-PD) was used at a concentration of 0 and 200 mg/kg/day for 10 days orally. The results showed that there was no cellular proliferation in the liver of male mice however in female mice 1.73 cellular proliferations in the liver were observed as compare to control. Therefore, it is considered that 4 -chloro-o-phenylenediamine (4-C-o-PD) in 200 mg/kg/day is negative for male mice and positive for female mice when mice are exposed to the test chemical for 10 days. However, In accordance with the CLP classification, the test material 4-chloro-o-phenylenediamine (4-C-o-PD) does not classify as a mutagen.

 

Based on the above mentioned in vitro and in vivo studies for target and read across substance and according to CLP criteria, it can be concluded that 2-chloro-p-phenylenediamine (CAS No. 615-66-7) is non genotoxic.

Justification for classification or non-classification

The results of several mutagenicity studies in vitro and in vivo shows that no classification for mutagenicity according to CLP regulation is warranted for 2-chloro-p-phenylenediamine (CAS No. 615-66-7).