2-nitro-p-phenylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

The test compound 2-nitro, p- phenylenediamine shows gene mutation in vitro and in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Micronucleus Test in Rats

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: unspecified

Vehicle:

0.5 percent (w/v) gum tragacanthConcentration of test material in vehicle: 2000 mg/kg

Details on exposure:

No data

Duration of treatment / exposure:

48 hours and 6 hours observation

Frequency of treatment:

Twice

Post exposure period:

6 hours

Remarks:

Doses / Concentrations:2000mg/kgBasis:no data

No. of animals per sex per dose:

5 rats/sex

Control animals:

not specified

Positive control(s):

no data

Tissues and cell types examined:

bone marrow smears were examined.

Details of tissue and slide preparation:

no data

Evaluation criteria:

no data

Statistics:

no data

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

RESULTS OF DEFINITIVE STUDYTypes of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No dataInduction of micronuclei (for Micronucleus assay): 2NPPD did not produce micronucleated polychromatic erythrocytes in ratsRatio of PCE/NCE (for Micronucleus assay): No dataAppropriateness of dose levels and route: No dataStatistical evaluation: No data

Conclusions:

Interpretation of results (migrated information): negativeTwo doses of 2000 mg/kg 2NPPD in 0.5 percent (w/v) gum tragacanth given 24 hours apart were administered orally to groups of 5 male and 5 female rats. The rats were killed 6 hours after the second dose, and bone marrow smears were examined. 2NPPD did not produce micronucleated polychromatic erythrocytes in rats

Executive summary:

Two doses of2000mg/kg2NPPD in 0.5 percent (w/v) gum tragacanth given 24 hours apart was administered orally to groups of 5 male and 5 female rats. The rats were killed 6 hours after the second dose, and bone marrow smears were examined.

2NPPD resulted in the production of orange urine. Agitation, convulsions, and lethargy were observed in animals.

2NPPD did notproduce micronucleated polychromatic erythrocytes in rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Gene mutation in vitro:

Various peer reviewed publications were studied to determine the mutagenic nature of the test compound 2- nitro-p- phenylenediamine (CAS no 5307 -14 -2). The summary is as mentioned below:

2-nitro-p-phenylenediamine (2NPD, CAS no 5307 -14 -2) is a direct-acting mutagen inSalmonella typhimuriumstrain TA100. The compound was tested further using the Xenometrix strains of S. typhimurium: TA7001 and TA7003, with and without S9 mix in the plate incorporation assay (Chung et al, 2000). Each of the strains detects only one unique base substitution event and only that event. The standard S. typhimurium plate incorporation assay was used in this study. Mixtures of cell suspension(100µl), sample solution(up to 100ml)the 0.1 M sodium phosphate buffer, and the Aroclor - induced rat liver S9_7%. _500ml.were added to the top agar and the mixtures were poured onto the bottom agar. All experiments were performed with triplicate plates at five doses. The slope of the dose–response curvethe number of revertants per microgram was calculated by least-squares linear regression from the first linear portion of the dose– response curve using the GeneTox Manager software. The data represent the mean±standard deviation of the numbers of revertant colonies from a final definitive experiment. The compound was non mutagenic to TA7001 and TA7003.

To determine the types of mutations involved, such as transversion or transition, the test compound2 nitro p phenylene diamine (2NPD, CAS no 5307 -14 -2) was tested with S. typhimurium strains TA4001 (Chen et al, 2000). Compound dissolved in DMSO were tested for dose-mutagenicity relationships using Salmonella typhimurium TA4001. The plate incorporation assay without metabolic activation was used. Each test dose was in triplicate with each tester strain. The experiments were done at least twice. The test compound was studied at a dose concentration of3, 10, 30, 100, 300, 1000 µg/plate. 2 nitro p phenylene diamine (2NPD) failed to induce reversion from TA to CG transition and hence isnot mutagenic in the S. typhimurium strain TA4001.

Gene mutation was predicted using SSS QSAR prediction model, 2016. The study used Salmonella typhimurium TA1535 strain and with S9 metabolic activation system. The test material 2-nitro-p-phenylenediamine is not mutagenic in vitro in Salmonella typhimurium strain TA 1535 without S9 metabolic activation system.

Gene mutation was predicted using SSS QSAR prediction model, 2016. The study used Salmonella typhimurium TA102 strain and without S9 metabolic activation system. The test material 2-nitro-p-phenylenediamine is not mutagenic in vitro in Salmonella typhimurium strain TA 102 without S9 metabolic activation system.

The mutagenicity of 2 -nitro, p-phenylenediamine (CAS no 5307-14-2) was tested using AmesSalmonellastrains TA98 and TA100 (Chung et al, 1995). The potency of the test compound in causing cytotoxic effects in CHO cells, as related by TC50 were determined. Mutagenicity tests were performed using the standard plate-incorporation and pre-incubation procedures. 2 - Aminofluorene, 2-nitrofluorene and MNNG were included as positive controls in all assays. The assays were performed in the presence or absence of the liver S9 mix. 2-Nitro p- phenylenediamine was mutagenic to strainsTA98 and TA 100 both in the presence and absence of the S9 mix, and greater mutagenic activity was observed with TA98 strain. Also at highest dose it was found to be toxic with and without metabolic activation.

 

The mutagenicity of 2 -nitro, p-phenylenediamine (CAS no 5307-14-2) was tested using chromosomal aberrations in Chinese hamster ovary (CHO) cells in the absence of the S9 mix (Chung et al, 1995). The potency of p-phenylenediamine and its derivatives in causing cytotoxic effects in CHO cells, as related by TC50 were determined. Chinese hamster ovary cells (CHO-Kl) were grown as monolayers in Dulbecco’s Modified Eagle medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum (FCS), and 1% antibiotic-antimycotic. The cultures were maintained at 37°C in a 5% CO2, humidified atmosphere. Mycoplasma-free cultures at about 1.5 x 10⁶cells per T75-cm²flask were maintained in 5 ml culture medium for 24 h before addition of test compounds. Exponentially growing cells (CHO-K1) in 60 x 15-mm culture dishes (Becton Dickinson & Co., Oxnard, CA) were incubated for 24 h with different concentrations of test chemicals in complete D-MEM/F-112 medium in order to establish the 50% toxic concentration (TC50) in this system. At the end of the exposure period, cultures were examined morphologically for cytopathic effects (CPE) using a phase-contrast microscope. At least 100 metaphases per flask were scored for each dose; individual types of aberration including breaks, deletions, exchanges and dicentrics were scored. Gaps were recorded but not included in the totals of aberrations. Both the percentage of aberrant cells (the number of cells with structural aberrations per 100 cells) and the frequency of aberrations (the total number of aberrations per 100 cells) were calculated. A positive dose-related increase in chromosomal aberration was observed with 2 nitro p-phenylenediamine. The analysis of individual aberrations from each dose showed that gaps, breaks, deletions, exchanges, and dicentrics were present. However, rings seldom occurred with 2-nitro-p-phenylenediamine.

 

2-nitro-p-phenylenediamine (2NPD, CAS no 5307-14-2) is a direct-acting mutagen in Salmonella typhimurium strain TA100. The compound was tested (Chung et al, 2000) further using the Xenometrix strains of S. typhimurium: TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006, with and without S9 mix in the plate incorporation assay (Chung et al, 2000). The standardS. Typhimuriumplate incorporation assay was performed. Mixtures of cell suspension(100µl), sample solution(up to 100ml)the 0.1 M sodium phosphate buffer, and the Aroclor - induced rat liver S9_7%. _500ml.were added to the top agar and the mixtures were poured onto the bottom agar. All experiments were performed with triplicate plates at five doses. The test compound was non mutagenic to TA7001 and TA7003. Mutagenicity of 2NPD was detected with TA7002, TA7004 and TA7005, indicating that the compound induces GC-AT transitions, and TA-AT and CG™AT transversions. In addition, 2NPD and DNBA showed some activity in TA7006, and DNBA showed activity in TA7003. 2NPD showed the greatest mutagenic response in strain TA7005.The second highest level of response for 2NPD was in TA7004.

 

Chromosomal aberration test (Ishidate et al, 1991) in vitro was performed for the test compound 2-nitro-p-phenylenediamine (CAS no 5307-14-2). Chromosome tests were carried out. CHL cells of about 10⁵in a monolayer culture in 6 cm plates were exposed too different doses of the test compound including the 50% growth inhibition dose . The cells were harvested at 24hr and 481hr after treatment and chromosome preparations were made according to the air-dry method The number of polyploid cells and cells with structural aberrations such as chromatid- type gaps ; breaks, exchanges, and rings;; was scored on 100 well-spread metaphases under a microscope For metabolic activation, a cell suspension of the CHL cells (10⁶/mf) was incubated together with the test agent and microsome fraction.(S9mix) which was prepared from the liver of Wistar rats pretreated with polychlorinated biphenyl (PCBs, KC-400) Chromosome preparations, were made after an additional culture for 24 hr in the same way as in the direct method. The final judgment was taken to be positive if the incidence of polyploid cells or cells with structural aberrations exceeded 10%, since the incidence in both untreated and solvent-treated. Control cells were usually less than 5%. For a quantitative comparison of the potential of chemical to introduce chromosomal aberrations (clastogenic activity), the value D20 was calculated, which shows the dose ( mg/ml) at which the aberrations were detected in 20% of metaphase cells . In addition, the TR-value was calculated from each chemical positive in the chromosome test, which indicates the incidence of cells with exchange type aberrations (translocation and ring, formation were included) per unit dose, mg/ml, of the test chemical.The results for chromosomal aberration test were reported in the form D20 and TR values.

2 nitro p phenylene diamine tested positive with and without S9 mix. The D20 value is 0.02mg/ml and TR value is 800mg/ml.

 

The reverse mutation assay (Ishidate et al, 1991) with strains TA98, TA100 and TA1537 were carried out according to the technique introduced by Ames combined with pre-incubation for 20 minutes before plating for the test compound 2-nitro-p-phenylenediamine (CAS no 5307-14-2). The final judgment was taken to be positive if the number of revertant colonies exceeded two times that in the untreated controls. 2 nitro p phenylene diamine was found to be mutagenic for the strain TA98.

 

Concentrations of 50-100 µg/ml 2NPPD caused chromosome and chromatid gaps and breaks in human peripheral blood lymphocytes cultures incubated for up to 72 hours (Cosmetoc Ingredients Review, 1985). A concentration of 100 µg/ml 2NPPD resulted in mitotic delay and toxicity, and 45 percent of the cells contained damaged chromosomes. Chromosome damage after incubation with lower concentrations of 2NPPD was not significantly different from the controls. 2 nitro p phenylenediamine can be considered to be mutagenic to human peripheral blood lymphocytes.

Gene mutation in vivo:

The study to determine the in vivo mutagenic nature of the test compound 2 -nitro, p- phenylenediamine (CAS no 5307 -14 -2) is summarized as below:

Two doses of 2000mg/kg 2NPPD (CAS no 5307 -14 -2) in 0.5 percent (w/v) gum tragacanth given 24 hours apart was administered orally to groups of 5 male and 5 female rats (Cosmetic Ingredients Review, 1985). The rats were killed 6 hours after the second dose, and bone marrow smears were examined. 2NPPD resulted in the production of orange urine. Agitation, convulsions, and lethargy were observed in animals.2NPPD did not produce micronucleated polychromatic erythrocytes in rats.

Justification for selection of genetic toxicity endpoint
The test compound 2-nitro, p- phenylenediamine is not mutagenic in vitro .

Justification for classification or non-classification

Based on the key studied summarized, the test compound 2 -nitro- p- phenylene diamine is not likely to classify as a gene mutant.