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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
A micronucleus test in bone marrow cells of the mouse should be conducted to clarify the relevance of positive findings from in vitro chromosome aberration tests (clastogenicity). This proceeding is in line with ECHA's Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7A: Endpoint specific guidance, version 6.0, July 2017 (Fig. R.7.7-1, flow chart of mutagenicity testing strategy). Due to the positive findings in the Ames test and the mammalian cell mutagenicity test, a 2nd in vivo test is required to clarify the relevance of these results (mutagenicity). A testing proposal for a transgenic rodent gene mutation assay is made in IUCLID section 7.6.2. The registrant proposes a tiered testing approach: First, the micronucleus test should be conducted. If this assay is positive and allows a clear conclusion regarding classification and labelling, the transgenic rodent gene mutation assay is obsolete and can be omitted. If the micronucleus test is negative, the transgenic rodent gene mutation assay should be performed as a second step to gain clarity regarding the mutagenic potential of the substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- N,N'-diphenyl-p-phenylenediamine (DPPD)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies: Three OECD TG and GLP studies were performed by the registrant that adress the genotoxic potential of DPPD. The substance was clearly mutagenic in the Ames test (BASF SE, 2016) and a mammalian cell gene mutation test in mouse lymphoma cells (BASF SE, 2017), and it induced structural aberrations in a chromosome aberration test (BASF SE, 2017). Evidence supporting these results is available from a GLP-compliant mammalian cell gene mutation test in mouse lymphoma cells published by US EPA (OTS0545453, 1987), where an increased mutant frequency of at least twice the mean mutant frequency in solvent controls was observed in the absence of metabolic activation. In a GLP-compliant published chromosome aberration test in CHO cells (US EPA, OTS0545526, 1988), a significant increase in chromosome aberrations was observed. Due to the statistically significant increase in structural aberrations at a single concentration at each of the harvest times (12 hour nonactivated study and 24 hour S-9 activated study) and the fact that at many of the concertation’s tested less than 100 cells were located for chromosome evaluation due to severe mitotic inhibition, the results from this study are considered equivocal.
While all of these studies are considered reliable without or only minor restrictions, they are in vitro tests and were performed in bacterial or mammalian cell culture. The relevance of these in vitro findings for the in vivo situation is not clear, and thus, they are not sufficient to evaluate the risk of the substance.

- Available non-GLP studies: Zeiger et al. (1992) reported that DPPD caused increased mutant frequencies in the Ames test in salmonella strains TA98 and TA100 in the presence of S9 mix. Sofuni et al. (1990) reported increased frequencies of aberrant cells in a chromosome aberration test in CHL cells without metabolic activation. No evidence of chromosomal aberration was found in CHO cells the same publication. Like in the available GLP studies mentioned above, the relevance of these in vitro findings for the in vivo situation is not clear, and thus, they are not sufficient to evaluate the risk of the substance.

- Historical human data: none available
- (Q)SAR: not applicable, in vitro guideline studies are available.
- In vitro methods: A number of high-quality in vitro tests for genetic toxicity is available that indicates a genotoxic potential of the substance (see above), but the relevance of these in vitro findings for the in vivo situation is not clear, and thus, they are not sufficient to evaluate the risk of the substance.
- Weight of evidence: The available in vitro data mentioned above can be evaluated in a weight-of-evidence approach and indicate a genotoxic potential, but the relevance of these in vitro findings for the in vivo situation is not clear, and thus, they are not sufficient to evaluate the risk of the substance.
- Grouping and read-across: not applicable, substance-specific information is available.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Annexes VI to X (and column 2 thereof) do not provide a possibility to waive an in vivo genotoxicity test when positive in vitro findings are available. On the contrary, section 8.4 in Annex VIII, column 2, states that "Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII."



Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-diphenyl-p-phenylenediamine
EC Number:
200-806-4
EC Name:
N,N'-diphenyl-p-phenylenediamine
Cas Number:
74-31-7
Molecular formula:
C18H16N2
IUPAC Name:
N,N'-diphenyl-p-phenylenediamine
Test material form:
solid

Test animals

Species:
mouse

Results and discussion

Applicant's summary and conclusion