Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
to be 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 : BLO (Blown Linseed oil) – Linseed oil, oxidised (CAS 68649-95-6 ; EC 272-038-8)

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: 5 in vitro studies on the target substance (OECD TG 471, OECD TG 473, negative and three OECD TG 476, 2 positive and 1 negative)

OECD Guideline 471 (Bacterial Reverse Mutation Assay) – negative, no indications of point mutations or frameshifts
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test) – negative, no indications for structural and numerical chromosome aberrations
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (2006) – positive (DMSO as solvent)
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (2012) – negative (acetone as solvent)
OECD Guideline 476 HPRT (In Vitro Mammalian Cell Gene Mutation Test) (2014) – mutagenic potential only to incubations without metabolic activation (acetone as solvent)

- Available non-GLP studies: none
- Historical human data: none
- (Q)SAR: not sufficiently reliable for the prediction of genetic toxicity
- In vitro methods: performed, and the study result provide sufficient base for further in vivo examination
- Weight of evidence: no data are available
- Grouping and read-across: A read-across is not sufficiently reliable for the prediction of this in vivo effect
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: 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:
The provision of an in vivo study is a standard REACH requirement. 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. It is not possible to meet this data requirement through any non-testing methods or any Column 2 or Annex XI adaptations.

• Neither in the bacterial mutagenicity test nor in the chromosomal aberration test in mammalian cells any indication for point or chromosomal mutagenic effects were noted.
• Three mammalian mutagenicity tests (mouse lymphoma assays) were performed.
o In one MLA the potential for a mutagenic potential at high, precipitating concentrations was noted.
o Concentrations showing no or only slight precipitation did not reveal any indication for a mutagenic potential in both tests, however a third MLA indicated mutagenic potential in the absence of metabolic activation.
o A further in-vivo test is foreseen to discard or confirm the genotoxic potential of Linseed Oil, Oxidized.


FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: For Linseed oil, Oxidized it is proposed by the registrant to perform an OECD TG 489 assay as a follow up study. This “In vivo alkaline single-cell gel electrophoresis assay for DNA strand breaks (Comet assay)” can provide conclusive information on both the clastogenicity as well as possible gene mutations of the test substance.


OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
in vivo mammalian cell study: DNA damage and/or repair

Principles of method if other than guideline
While the test item Linseed oil, oxidized did not induce gene mutations in bacteria (Ames test, OECD 471) or chromosome aberrations in mammalian cells (Chromosome Aberration Assay, OECD 473), positive and negative outcomes of point mutations were observed in mammalian cells (Mouse Lymphoma Assay and HPRT Assay, OECD 476).
According to REACH requirements Annex VIII/IX, section 8.4 an appropriate in vivo gene mutation test shall be proposed by the registrant, when there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD 471) or from an in vitro gene mutation study in mammalian cells (OECD 476).

Based on the available in vitro data, an alkaline in vivo Comet assay (OECD 489) is proposed to assess the mutagenic properties of the test substance in vivo. The in vivo Comet assay is considered to be the appropriate test system to investigate short-lived substances at the first site of contact. Furthermore, historical control data are available for various tissues. The test substance is proposed to be administered orally by gavage to rats. Doses will be based on data available for repeated dose toxicity and acute oral toxicity study. Based on the results of the repeated dose toxicity and acute oral toxicity study and taken the OECD requirements into consideration, a maximum dose of 2000 mg/kg bw/day, and two additional doses are proposed. Organs proposed to be evaluated are the forestomach and the liver, as the site of first contact after oral administration and the site of metabolism, respectively. The tissues are proposed to be evaluated after two administration days.
Six animals in the dose groups and negative control group as well as 4 animals in the positive control groups are proposed to reach a minimum of 5 and 3 analysable animals per sex, respectively, as requested according to the Guideline. Since the available data did not demonstrate relevant differences between males and females, the use of males is proposed.

Type of assay
single cell gel/comet assay in rodents for detection of DNA damage
mammalian cell study: DNA damage and/or repair

Species
Rat

Route of administration
oral: gavage

Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
current
Deviations:
no

Test material

Constituent 1
Reference substance name:
Linseed Oil, oxidised
IUPAC Name:
Linseed Oil, oxidised
Constituent 2
Reference substance name:
Linseed oil, oxidized
EC Number:
272-038-8
EC Name:
Linseed oil, oxidized
Cas Number:
68649-95-6
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Linseed oil, oxidized
Test material form:
other: kight yellow liquid
Details on test material:
- Name of test material (as cited in study report): Linseed Oil, oxidized
- Physical state: light yellow liquid
- Water solubility: < 0.001 g/L
- Soluble/Emulsifiable in acetone at 406 mg/ml
- Storage requirements: room temperature

Results and discussion

Applicant's summary and conclusion