Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: 1-imino-1H-isoindol-3-amine (Ingrain Blue 2.2)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies - in vitro methods:
1) Bacterial reverse mutation assay (Ames) according to OECD 471: The outcome of this study was negative, i.e. the test substance is not mutagenic towards bacteria.
2) Micronucleus study according to OECD 487: The outcome of this study is negative, i.e. the test item did not show clastogenic and/or aneugenic activity.
3) Mammalian cell gene mutation study, using thymidine kinase gene (mouse lymphoma) according to OECD 490: The result of this study is positive, i.e. clastogenic effects were induced by the test item.
- Weight of evidence: Since at least one positive result was observed, further in vivo testing is needed.
- Available non-GLP studies: not available
- Historical human data: not available
- (Q)SAR: not available
- Grouping and read-across: no read-across candidates identified.
- Substance-tailored exposure driven testing: not applicable


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:
All possible adaptation possibilities were considered and none were applicable. Exposure-based waiving could also not be used.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
To gain further insight on the genetic toxicity potential of the test item, we propose to perform an in vivo comet assay (OECD TG 489) on intestinal cells which is an indicator test for DNA lesions.

As systemic exposure cannot be guaranteed from the acute and subacute toxicity studies, and no toxicokinetic data are available, an in vivo Micronucleus assay is not proposed as this bears the risk that the substance will not reach the bone marrow. Instead an in vivo Comet assay (OECD TG 489) is proposed with liver and small intestine (duodenum or jejunum, depending on experience of lab) as target tissues.

The liver is the standard organ of the Comet assay, but if the substance is not absorbed, the liver will not be reached. Therefore it is proposed to add gastro-intestinal tissue as target organ. The stomach is usually first site of contact for substances after oral exposure, although other areas of the gastro- intestinal tract such as the duodenum and jejunum can also be considered as site-of-contact tissues and may be considered more relevant for humans than the rodent glandular stomach. Care should be taken to ensure that such tissues are not exposed to excessively high test substance concentrations, but rather to expected exposure concentrations to avoid false positive reactions (Donovan & Burlinson, 2013).

Reference:
Donovan MO & Burlinson B. Maximum dose levels for the rodent comet assay to examine damage at the site of contact or to the gastrointestinal tract. Mutagenesis vol. 28 no. 6 pp. 621–623, Advance Access publication 3 October 2013;

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
yes
Remarks:
with liver and small intestine (duodenum or jejunum, depending on experience of lab) as target tissues.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Results and discussion

Applicant's summary and conclusion