Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Safepharm, 2005

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

BioReliance, 2000

Ames Test

(OECD 471)

S, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

TA 102

E.coli WP2 uvrA

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

Harlan, Woods 2015

CHO/HPRT test (OECD 476)

K, rel. 2

Gene mutation

Chinese hamster ovary (CHO)cells

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

4 (Read Across)

 

BASF, 2003

In vivo MN

(OECD 474)

K, rel. 2

Chromosomal aberration

Mice

 -

Up to toxic concentrations

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -3):

- Two Bacterial Reverse mutation Assays (Ames tests) were performed according to OECD guideline No. 471 with the substance (Test n°1 & 2, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation, in both assays. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in Chinese hamster CHO cells (CHO/HPRT test) (Test n°3). None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments. The substance does not induce forward mutations at the HPRT locus in CHO chinese hamster cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore the registered substance is considered as negative for inducing gene mutations at the HPRT locus in CHO chinese hamster cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

Chromosomal aberration (Test n°4)

The clastogenic and aneugenic potential of the supporting substance, (E)-4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-3-buten-2-one, which is considered adequate for read-across purpose (see Iuclid section 13 for additional justification), was determined using an in vivo mammalian erythrocytes micronucleus assay (Test n°4), which identifies substances that cause micronuclei in erythroblasts. These micronuclei may originate from acentric fragments or whole chromosomes, and the test thus has the potential to identify both clastogenic and aneugenic chemicals. In this study erythroblasts were sampled from bone marrow cells of mice. None of the i.p. dose levels of up to 750 mg/kg bw, in males, induced increase in the frequency of micronucleated polychromatic erythrocytes (fMPCE), whereas the positive control chemical induced significant increases in the fMPCE. The supporting substance was therefore considered as negative for inducing chromosomal aberrations in mice bone marrow erythrocytes under the conditions used in this study. The supporting substance, and by analogy the registered substance, are therefore considered as non-clastogenic and non-aneugenic.


Justification for selection of genetic toxicity endpoint
No study was selected, since all in vitro and in vivo studies performed on the substance itself or on a supporting substance were negative and of high quality.

Short description of key information:
- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- Ames Test (OECD 471, GLP, S, rel. 1): non mutagenic up to limit concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- CHO/HPRT Mammalian Cell Gene Mutation Assay (OECD 476, GLP, K, rel. 2): non mutagenic at any dose level.
- In vivo Bone marrow micronucleus test (OECD 474, GLP, Read-Across, K, rel. 2): non clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including ATP6.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).