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No study was available on the substance itself to assess genotoxicity potential, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

The registered substance is present at 10% in the mixture, i.e. well above the concentration triggering classification of mixture as germ cell mutagens as defined in the Annex VI of the Regulation (EC) No. 1272/2008, therefore it can be safely concluded that the pure cis-isomer does not need to be classified as a mutagen.

Table 7.6.1/1: Summary of Genetic Toxicity Tests conducted on the supporting substance:

Test n°

Test guideline / reliability

Test Substance isomer composition

Focus

Strains tested

Metabolic activation

Test concentration (µg/plate or mL)

Statement

1

Ames Test (OECD 471)

K, rel.2

90% trans-

10%cis-

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

TA 102

-S9

+S9

Up to 5000

-S9 : non mutagenic

+S9 : non mutagenic

2

ML/TK test (OECD 476)

K, rel.2

90% trans-

10%cis-

Gene mutation

mouse lymphoma L5178Y cells

-S9

+S9

Up to 250

-S9 : non mutagenic

+S9 : non mutagenic

3

Mammalian Erythrocyte Micronucleus Test

(OECD 474)

K, rel.2

90% trans-

10%cis-

Chromosomal aberration

mouse  

(intraperitoneal)

N.A.

Up to 1120 mg/kg

Non-mutagenic

4

UDS Assay (OECD 486)

K, rel.2

90% trans-

10%cis-

DNA damage repaired by unscheduled DNA synthesis

rat

N.A.

Up to 1000 mg/kg

(intraperitoneal)

Non-Genotoxic

Gene mutation Assays (Tests n° 1-2):

A Bacterial Reverse mutation Assay(Ames test)was performed on the supporting substance according to OECD 471 test guidelines with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test indicates that 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 mammals using anin vitroreverse mutation assay in mouse lymphoma TK L5178Y cells(ML/TK test)(Test n°2). None of the treatment up to the cytotoxicity limits with the substance, with or without metabolic activation, induced significant mutant frequency increases in initial, repeat and confirmatory tests. The substance does not induce forward mutations at the TK locus in L5l78Y mouse lymphoma cells under activation and non-activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases.The substance is therefore considered as negative for inducing forward mutations at the TK locus in L5l78Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of both Ames tests and extends the non-mutagenic effect of the test substance to mammalian cells.

 

Chromosomal aberration (test n°3)

An in vitro chromosome aberration test has not been performed on the supporting substance. However, in test No. 2 (mouse lymphoma assay, MLA) the mutant colonies were assessed as being large or small. Small colonies have been shown to be primarily the result of clastogenic damage to chromosome 11 (the site of the TK gene). Consequently, an MLA assay including the assessment of small colony frequency is considered to be a surrogate for an in vitro chromosome aberration test. Furthermore, the clastogenic potential of the supporting substance was determined using anin vivoMammalian Erythrocyte Micronucleus Test which measures the potential of a substance to increase the incidence the of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. At least 5 males and 5 females mice were treated (ip route) at constant volumes in corn oil for the lowest treatment concentration and cyclophosphamide (50mg/kg) positive control (280, 560 mg/kg), 15 mice for 1120 mg/kg and then mice for the mock. Doses were selected from a toxicity assay: the highest dose for the micronucleus correspond to ca. 80% of the LD50. Mortality was observed in 4/15 male and 1/15 female mice dosed with 1120 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. No significant increase in micronucleated polychromatic erythrocytes was observed in either male or female mice (p > 0.05), whereas positive control (cyclophosphamide) provided significant increases (p ≤ 0.05).The test substance is considered as non-mutagenic according to the OECD TG 474Mammalian Erythrocyte Micronucleus Test.

 

DNA damage repaired by unscheduled DNA synthesis (test n°4)

The ability of the supporting substance to induce DNA damage (i.e. patch repair) in vivo was assessed using an UDS Assay (Test n°5) in isolated rat hepatocytes following administration of the substance to rats. Intraperitoneal route was selected as preliminary toxicity tests indicated that no toxicity occurred at 2000 mg/kg (oral) whereas evidence of systemic absorption and toxicity occurred both in male or female rats treated intraperitoneally. As both gender were similarly affected, only males were used for the main study. Four rats were used for the both test concentration (333 and 1000 mg/kg) and for both positive controls whereas six rats were used for the negative control (corn oil). The study was performed in two parts, in experiment 1 the livers were perfused approximately 16 hours after dosing and, in experiment 2, perfusion was performed approximately 2 hours after dosing. Further groups of rats were given a single intraperitoneal dose of arachis oil, or dosed orally with 2-acetylaminofluorene (2-AAF) at 16 hours orN,N'-dimethylhydrazinedihydrochloride (NDHC) at 2 hours to serve as vehicle and positive controls respectively.

There wasno marked increase in the incidence of unscheduled DNA synthesis in animals dosed with the test materialat either time point. The positive controls both produced marked increases in the incidence of cells in repair. The test material was considered therefore to be non-genotoxic under the conditions of the test.

 

CONCLUSIONS:

All the tests performed are complementary as they focus on different kind of damage a chemical or its metabolites can induce to DNA. This ensemble of studies investigating gene mutation both in bacteria and mammals, mammalian chromosome aberration and mammalian DNA damage repaired by unscheduled DNA synthesis process, provide evidence thatthe supporting substance (and the registered substance) has no genotoxicology concerns.


Justification for selection of genetic toxicity endpoint
No study was available on the substance itself, therefore a read-across approach was used. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification). No study was selected, since all in vitro studies performed on the supporting substance, were negative.

Short description of key information:
1. Ames Test, Strains: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & TA 102 tested both with and without metabolic activation (S9 from rat): non mutagenic up to 5000 µg/l (OECD 471, GLP, read-across, Rel.2)
2. ML/TK Forward Mutation Assay with a Confirmatory Assay, Mouse Lymphoma TK L5178Y tested both with and without metabolic activation: non mutagenic up to cytotoxicity limit (OECD 476, GLP, read-across, Rel.2)
3. Mammalian Erythrocyte Micronucleus Test (intraperitoneal), mouse, no significant increase in micronucleated polychromatic erythrocytes in either male or female mice up to toxicity limit (80% of intraperitoneal LD50): non mutagenic. (OECD 474, GLP, read-across, Rel.2)
4. Unscheduled DNA Synthesis (UDS) Assay, rat, non genotoxic up to 1000 mg/kg (intraperitoneal) (OECD 486, GLP, read-across, Rel.2)
 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including ATP4.

Self-classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

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