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Key value for chemical safety assessment

Additional information

Table1: Genetic Toxicity Test performed:

Test n°

Test guideline

Focus

Strains tested

Metabolic activation

Test concentration (µg/l)

Statement

1

Ames Test (OECD 471)

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

TA 102

-S9

+S9

Up to 5000

-S9 : non mutagenic

+S9 : non mutagenic

2

Ames Test (OECD 471)

Gene mutation

TA 1535,

TA 1537

TA 98

TA 100

WP2 uvrA

-S9

+S9

Up to 5000

-S9 : non mutagenic

+S9 : non mutagenic

3

ML/TK test (OECD 476)

Gene mutation

mouse lymphoma L5178Y cells

-S9

+S9

Up to 250

-S9 : non mutagenic

+S9 : non mutagenic

4

Mammalian Erythrocyte Micronucleus Test (OECD 474)

Chromosomal aberration

mouse  

(intraperitoneal)

N.A.

Up to 1120 mg/kg

Non-mutagenic

5

UDS Assay (OECD 486)

DNA damage repaired by unscheduled DNA synthesis

rat

N.A.

Up to 1000 mg/kg

(intraperitoneal)

Non-Genotoxic

 

ML/TK test: Mouse Lymphoma Forward Mutation Assay with a Confirmatory Assay

 

Gene mutation Assays (Tests n° 1-3):

Two Bacterial Reverse mutation Assays (Ames test) were performed according to OECD 471 test guidelines with the substance (See Table 1). Test n°1 was selected as key study as in test n°2, 2-Aminoanthracene was used as sole indicator of the efficacy of the S9-mix which did not provide a positive control of mutagenicity due to microsomal activation. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains in both tests, with any dose of the test material, either with or without metabolic activation. Both tests indicate 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°3). 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 notinduce forward mutations at the TK locus in L5l78Y mouse lymphoma cellsunder activation and nonactivation conditionswhereas 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°4)

Clastogenic potential of the substance was determined using an in vivo Mammalian 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 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 474 Mammalian Erythrocyte Micronucleus Test.

 

DNA damage repaired by unscheduled DNA synthesis (test n°5)

The ability of the 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 was no marked increase in the incidence of unscheduled DNA synthesis in animals dosed with the test material at 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. Therefore, there is no “key study” selected excepted for studies having the same endpoint. 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 that the substance has no genotoxicology concerns.



Short description of key information:
1. Ames Test (OECD 471), 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
2. Ames Test (OECD 471), Strains: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.Coli WP2 uvrA tested both with and without metabolic activation (S9 from rat): non mutagenic up to 5000 µg/l
3. ML/TK Forward Mutation Assay with a Confirmatory Assay (OECD 476), Mouse Lymphoma TK L5178Y tested both with and without metabolic activation: non mutagenic up to cytotoxicity limit
4. Mammalian Erythrocyte Micronucleus Test (intraperitoneal) (OECD 474), mouse, nosignificant increase in micronucleated polychromatic erythrocytes in either male or female mice up to toxicity limit (80% of intraperitoneal LD50): non mutagenic.
5. Unschedulded DNA Synthesis (UDS) Assay (OECD 486), rat, non genotoxic up to 1000 mg/kg (intraperitoneal)
 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

As the substance has no mutagenic and genotoxic concerns, there is no mutagenicity and genotoxicity classification.