2-chloro-p-phenylenediamine

Endpoint summary

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 or test system 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).