Reactive Orange 16

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

other: read-across from supporting 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:

guideline study

Justification for type of information:

NON-CONFIDENTIAL NAME OF SUBSTANCE:

Name of the substance on which the test has been performed: refer to the attached document

This test will be used in read-across on the target substance Reactive Orange 16 (EC: 701-348-2).


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

a) This part refers to the available studies on the target substance Reactive Orange 16 (EC: 701-348-2):

- 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:

b) This part refers to the available studies on the analogue substance that were used in read across to cover the endpoint of genotoxicity of Reactive Orange 16 (EC: 701-348-2):
In vivo alkaline Comet Assay (OECD 489)
 

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, the performance of an appropriate in vivo somatic cell genotoxicity study must be considered if there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII.
Guidance on information requirements R7a, section 7.7.6 (2017), states that regarding Annex VIII, when both the 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 choice of the appropriate in vivo study is triggered by the availability of reliable cytogenicity studies, either in vitro or in vivo, covering the endpoint. In fact,
a. If the test results of the available cytogenicity test(s) are negative, an in vivo mammalian alkaline comet assay (test method: OECD TG 489) in rats, or if justified, in other rodent species, oral route, on the following tissues, liver, glandular stomach and duodenum needs to be performed
b. If the test results of the available cytogenicity test(s) are positive, an in vivo mammalian alkaline comet assay (test method: OECD TG 489) combined with in vivo mammalian erythrocyte micronucleus test (test method: OECD TG 474) in rats, or if justified, in other rodent species, oral route needs to be performed. For the In vivo mammalian alkaline comet assay the following tissues shall be analysed: liver, glandular stomach and duodenum
The submitted dossier Reactive Orange 16 EC 701-348-2 contains results for the in vitro gene mutation study in bacteria, following OECD 471, which raises the concern for in vivo gene mutation, since it shows positive results (i.e., an increment of the number of revertants for strain WP2 uvrA with S9 mix at concentrations > 1000 ug/plate with Prival modification and pre-incubation).
An in vitro mammalian chromosome aberration test OECD 473, performed on an analogue substance is inserted, which was considered to fulfill the information requirement. The tested substance resulted non-clastogenic to cultured human peripheral blood lymphocytes, under the experimental conditions. Moreover, an in vivo chromosomal aberration OECD 475 on the same analogue substance, is submitted and the substance resulted not mutagenic in bone marrow cells of the Chinese hamster under the experimental conditions. Based on the considerations reported in the read across justification, same results can be applied also to Reactive Orange 16 and cytogenicity can be considered overall negative.
Therefore, by summarizing all the above available information, an in vivo mammalian alkaline Comet Assay needs to be performed on analogue substance 3 in order to elucidate the in vivo gene mutation potential. The information requirements is fulfilled by applying read across approach from analogue substance 3 , for which an in vivo Comet Assay testing proposal was accepted and will be performed, to target substance Reactive Orange 16.


Both target and analogue substance show positivity to the OECD471 test. Both substances rely for cytogenicity to analogue substance 2, which, as reported in the read across section, in particular based on the submitted toxicokinetics studies, can be used for predicting this genotoxicity potential. Meanwhile, an in vivo alkaline Comet Assay, OECD 489, was requested by the Authorities involving stomach, liver and duodenum as target organs for analogue substance 3.
Altogether the above information on genotoxicity and the considerations reported in the read across document, justify the use of the results of the OECD489 for assessing the potential in vivo gene mutation properties of the target substance Reactive Orange 16.

OECD 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 recognizes 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.

CONCLUSIONS
In order to completely assess the in vivo genotoxic potential of Reactive Orange 16, triggered by positive results in an vitro gene mutation study in bacteria and negative results in an in vitro cytogenicity test, a Comet Assay performed on analogue substance is used in read across. The two substances share chemical similarities, phys-chem and toxicological properties that allow the use of the read across methodology.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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:

Similar substance 3

IUPAC Name:

Similar substance 3

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

No. of animals per sex per dose:

5 males/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

yes

Results and discussion

Test results

Sex:

male

Genotoxicity:

other: in progress

Remarks on result:

other: the test is in read-across from an on going OECD 489 on a similar substance

Applicant's summary and conclusion

Conclusions:

study in progress