[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read across from supproting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

not yet defined

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:

NON-CONFIDENTIAL NAME OF THE SUBSTANCE:
Name of the substance on which testing is proposed to be carried out: analogue substance 03

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
Available GLP studies: not available.
Available non-GLP studies: not available.
Historical human data: not available.
(Q)SAR: not available.
Weight of evidence: not available.
Grouping and read-across:
- OECD TG 476 (In Vitro Mammalian Cell Gene Mutation Test) – Key study – Read-Across on analogue substance 02.
- OECD TG 471 – NR-deficient strains – Key study – Read-Across on analogue substance 02.
- OECD TG 471 (IHMA) – Supporting study – Read-Across on analogue substance 02.
- OECD TG 471 – Only strain TA 100 – Disregarded study – Read-Across on analogue substance 02.
- OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) – Key study – Read-Across on analogue substance 02.
- OECD TG 486 (Unscheduled DNA Synthesis) – Key study – Read-Across on analogue substance 02.

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:
Under Annex VIII Section 8.4., column 2 of REACH, further mutagenicity studies must be considered in case of a positive result in an in vitro gene mutation study in bacteria.

Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both mammalian cell tests are negative but there was a positive result in the bacterial test, it will be necessary to decide whether any further testing is needed on a case-by-case basis. For example, suspicion that a unique positive response observed in the bacterial test was due to a specific bacterial metabolism of the test substance could be explored further by investigation in vitro. Alternatively, an in vivo test may be required.

The submitted dossier on Acid Brown 332:1 contains positive results for the in vitro gene mutation study in bacteria, following OECD TG 471 on the analogue substance 02, which raises the concern for gene mutation.

As presented in the attached document regarding genotoxicity, a fist attempt to explain the mechanism of the nitroreductase in the positivity of the Ames test was evaluated on the same read across substance [analogue substance 02]. The test substance was tested in a modified Ames test following OECD TG 471 with NR deficient strains, namely TA 98 and TA 100, using the Prival method. Results really support the major role played by the nitroreductase and a great decrease on the number of revertants with respect to the control was obtained for TA 98NR and TA 100NR. However, the modified Ames test cannot be finalised as totally negative.
Annex VIII, Column 2, requires the registrant to consider appropriate mutagenicity in vivo studies already at the Annex VIII tonnage level, in cases where positive results in genotoxicity studies have been obtained, which involves studies mentioned in Annex IX (as first step OECD TG 474 – Mammalian Erythrocyte Micronucleus Test, OECD TG 488 – Transgenic Rodent Mutation Assay, OECD TG 489 – In Vivo Mammalian Alkaline Comet Assay and OECD TG 486 – Unscheduled DNA Synthesis).

CONSIDERATIONS ON THE STUDIES INSERTED IN THE PRESENT DOSSIER AND EXPERT ASSESSMENT ON TESTING PROPOSAL:
The submitted dossier on Acid Brown 332:1 contains an OECD TG 474 (Mammalian Erythrocyte Micronucleus Test) in vivo study, conducted on the analogue substance 02, with negative results, which is adequate to cover the chromosomal aberration potential of the two substances and to waive the performance of an in vitro cytogenicity in mammalian cells, as laid down in Column II of Annex VIII of the REACH Regulation.
Moreover, an OECD TG 486 (in vivo UDS assay) is also present and conducted in read across on the analogue substance 02, which resulted negative and can be used as supporting information for the gene mutation properties, since cells analysed in the UDS assay involve only those of the liver.

However, in order to further and completely assess the gene mutation properties of the substance in different tissues of the animal, a Comet Assay, OECD TG 489, on the analogue substance 03 is presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of Acid Brown 332:1.

Analogue substance 03 is, in fact, considered as representative of the mutagenic behaviour of Acid Brown 332:1 in a worst-case scenario approach, showing a positivity also in the in vitro gene mutation test.

OECD TG 489 allows to measure DNA strand breaks, that may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.

OECD TG 488 is not considered the first choice for assessing the gene mutation in vivo for this substance, since preliminary data for gene mutation in vivo (OECD TG 486) already indicate negativity in the somatic cells of the liver. A confirmation by the Comet assay performed over other tissues (and for azo dyes the intestinal tract is the site of major metabolism and dye/metabolites absorption) would be sufficient to assess the genotoxic potential of the substance.

Finally, as reported in literature, from the analysis of 91 chemicals with published data from Comet Assay and Transgenic rodent mutation assay (TGR), the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract (predominantly stomach and colon data) and, hence, can be confidently performed to confirm in vivo gene mutation activity in terms of genotoxicity in general.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

Analogue Substance 03

IUPAC Name:

Analogue Substance 03

Test material form:

solid: particulate/powder

Administration / exposure

Route of administration:

oral: gavage

Results and discussion

Test results

Sex:

not specified

Genotoxicity:

other: to be performed

Remarks on result:

other: the test is in read across from a submitted testing proposal still under evaluation