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Key value for chemical safety assessment

Additional information

Ames Tests:

All bacterial and yeast tests are conducted before 1997, when the actual OECD TG 471 was adopted.

Rannung (1984): Genotoxic effects of additives in synthetic elastomers with special consideration to the mechanism of action of thiurames and dithiocarbamates

4 strains: TA98, TA100, TA1535, TA1537

Result: negative (with and without metabolic activation)

Crebelli: (different studies are summarized conducted 1982-1984; studies are conducted according to Ames 1975; limited documentation)

5 strains: TA98, TA100, TA1535, TA1537, TA1538

Result: negative (with and without metabolic activation)

2 strains: TA98, TA100

Result: negative (with and without metabolic activation)

Flowers (1983): Ames/Salmonella mutagenicity assays of Flectol H and Flectol Pastilles, Report No.: MSL-2969

2 Strains: TA98, TA100

Result: negative (with and without metabolic activation)

Yamaguchi (1991): Mutagenicity of Rubber Additives in Tire,

2 Strains: TA98, TA100

Result: negative (with and without metabolic activation)

Brusick (1976): Mutagenicity evaluation of Bio-76-109 Flectol H, Report no.: LBO Project no. 2547

5 strains: S.typhimurium TA98, TA100, TA1535, TA1537, TA1538 and Saccharomyces cerevisiae strain D4

Result: negative (with and without metabolic activation)

Brusick (1977): Mutagenicity Evaluation of Flectol Flakes Fraction 5 BIO-77-143e ERL 20389/20-5

5 strains: S.typhimurium TA98, TA100, TA1535, TA1537, TA1538 and Saccharomyces cerevisiae strain D4

Result: negative (with and without metabolic activation)

Tests on mammalian cells

Cytogenetic Tests:

Hall (2010), In vitro chromosome aberration test in Chinese hamster V79 cells with 1,2-dihydro-2,2,4-trimethylquinoline, oligomers

Result: negative (with and without metabolic activation)

Li (1986): In vitro cytogenetics study of Flectol H (chromosome aberration test), Report no. MSL-6067

Chinese hamster ovary (CHO) cells

Result: negative (with and without metabolic activation)

Unscheduled DNA sysnthesis:

Mirsalis (1984): Evaluation of the potential of Flectol H antioxidant to induce unscheduled DNA synthesis in primary rat hepatocyte cultures, Monsanto study no. SR-83-285

Result: negative

Gene-mutation tests:

Hall (2010), Gene mutation assay in Chinese Hamster V79 cells in vitro (V79/HPRT) with 1,2-Dihydro-2.2.4-Trimethylquinoline, oligomers

Result: negative (with and without metabolic activation)

Godek (1985): CHO/HGPRT Mammalian cell forward gene mutation assay, PH 314-MO-009-83, Flectol H antioxidant, study number

Result: negative (with and without metabolic activation)

Brusick (1979): Mutagenicity evaluation of Flectol flakes in the mouse lymphoma forward mutation assay, LBI project no. 20989

L5178Y mouse lymphoma cells

Result: negative (with and without metabolic activation)

Myhr (1979), Mutagenicity Evaluation of Flectol H (BO-78-237) in the Mouse Lymphoma Forward Mutation Assay

L5178Y mouse lymphoma cells

Result: negative (with and without metabolic activation)

Results and discussion:

Ames tests:

1,2-dihydro-2,2,4-trimethylquinoline, oligomers (which is an antioxidant with no crosslinking properties and not a hydrazine(derivative)) was tested in several Ames tests including the Salomonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Saccharomyces cerevisiae strain D4 with and without metabolic activation.

In all tests, 1,2-dihydro-2,2,4-trimethylquinoline, oligomers was negative.

Partly the assays were not conducted according actual OECD guidelines or according GLP.

Nevertheless, the results of all 6 assays are considered as reliable, as all tests have the same result (negative – with and without metabolic activation).

Cytogenetic assays:

1,2-dihydro-2,2,4-trimethylquinoline, oligomers (which is an antioxidant with no crosslinking properties and not a hydrazine(derivative)) was further tested in several cytogenetic assays.

In the in-vitro mammalian chromosome aberration test by Hall (2010) conducted according to OECD TG 473 and according to GLP, TMQ did not induce structural chromosome aberrations in V79 cells in vitro and consequently, the test item is not clastogen in the presence and absence of metabolic activation when tested up to cytotoxic and/or precipitating concentrations.

Result: negative (with and without metabolic activation)

In the Study by Li (1986) Flectol H was tested for its potential to induce chromosomal aberrations in cultured Chinese hamster ovary cells. CHO cells were treated with 50, 100, and 200 ug/ml of Flectol H for 5 hrs in the absence and presence of exogenous activation. No significant increases in chromosomal aberrations were observed in the cells at any of the time points or dose levels of Flectol H tested.

Result: negative (with and without metabolic activation)

Unscheduled DNA synthesis

Additionally, the ability of FLECTOL H pastilles to induce unscheduled DNA synthesis (UDS) in the in vitro hepatoeyte DNA repair assay was evaluated by Mirsalis (1984). This indicator test investigated the induction of the DNA repair system in mammalian cells in response to genetic effects induced by a chemical. The results indicate that FLECTOL H Antioxidant is not a genotoxic agent in the in vitro rat hepatocyte DNA repair assay.

Result: negative

Mammalian cell gene mutation tests:

In a Gene mutation assay in Chinese Hamster V79 cells in vitro (V79/HPRT) with 1,2-Dihydro-2.2.4-Trimethylquinoline, oligomers by Hall (2010), no substantial and reproducible dose dependent increase in mutation frequency was observed in both experiments in the absence or in the presence of metabolic activator S9. 1,2 -Dihydro-2,2,4 -trimethylquinoline, oligomers is considered as non-mutagenic in this HPRT assay.

Result: negative (with and without metabolic activation)

In the CHO/HGPRT Mammalian Cell Forward Gene Mutation Assay by Godek (1985), Flectol Pastilles, were negative in the in the assay, with and without metabolic activation.

Result: negative (with and without metabolic activation)

The test compound, Flectol H (B0-78-237), did not induce an increase in mutations at the TK locus in L5178Y mouse lymphoma cells over the dose ranges of 62 .5 to 750 µg/ml without activation and 250 to 3000 µg/ml with S-9 microsomal activation (Myhr, 1979).

Result: negative (with and without metabolic activation)

In another mouse lymphoma assay, the test compounds BIO-76 -250 CP 4058 and Flectol flakes were evaluated in the L5178Y cell line.

BIO-76-250 CP 4058 (Flectol H Powder), did not induce gene mutation at the TK locus in L5178Y cells, with and without metabolic activation.

The test material, Flectol Flakes, Lot #9C967, did not induce an increase in the mutant frequency at the TK locus in L5178Y mouse

Result: negative (with and without metabolic activation)

Conclusion:

In 6 Ames tests on S. typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) and Saccharomyces D4, all tests were negative (with and without metabolic activation). By a WoE consideration, TMQ (1,2-Dihydro-2.2.4-Trimethylquinoline, oligomers) is negative in microbial assays.

In the OECD test guideline # 471, the recommended combinations of strains included S.typhimurium 102 or E.coli WP2 uvrA or E. coli WPS uvrA (pKM101) because these strains may detect certain oxidizing mutagens, cross-linking agents and hydrazines.

1,2-dihydro-2,2,4-trimethylquinoline, oligomers is an antioxidant with no crosslinking properties and not a hydrazine(derivative).

Due to these intrinsic chemical properties of 1,2-dihydro-2,2,4-trimethylquinoline, oligomers an additional Ames test seems not justified based on scientific reasons. The requirement of OECD TG # 471 is regarded as fulfilled.

As described in the OECD TG # 471, in order to detect cross-linking mutagens it may be preferable to include TA102 or to add DNA repair proficient strain of E.coli [e.g. E.coli WP2 uvrA or E. coli WPS uvrA (pKM101)]

For the detection of DNA damage and repair or unscheduled DNA synthesis in mammalian cells in vitro a valid UDS (unscheduled DNA synthesis) test and 2 valid chromosome aberration tests are available. All 3 tests were negative.

A DNA damage or repair was not detected by these cytogenetic assays and there is no indication for cross-linking properties of 1,2-dihydro-2,2,4-trimethylquinoline, oligomers. Additional four valid mammalian cell gene mutation assays (HPRT tests) in V79 and CHO cell lines of Chinese hamster cells and L5178Y mouse lymphoma cells are available.

These higher tier assays which are able to detect a broad spectrum of mutagens were all negative with and without metabolic activation.

Overall:

1,2-dihydro-2,2,4-trimethylquinolone, oligomers, CAS no 26780-96-1 is used as an antioxidant, has no crosslinking properties and is not a hydrazine/hydrazine derivative. Consequently, the chemical structure and properties do not trigger additional testing in E.coli WP2 or S. typhimurium TA 102.

Based on the six negative Ames tests including different bacteria and yeast strains, the two negative chromosome aberration tests in mammalian cells, the negative UDS test in mammalian cells and the 4 negative HPRT tests in mammailian cells, the genotoxicity potential of 1,2-dihydro-2,2,4-trimethylquinoline, oligomers is well examined. Not a single positive in vitro genotoxicity test is reported.

Therefore, an additional Ames test with S.typhimurium 102 or E.coli WP2 uvrA or E. coli WP2 uvrA (pKM101) is not justified taking into accound the chemical properties and the available comprehensive genotoxicity data.


Justification for selection of genetic toxicity endpoint
Based on the six negative Ames tests including different bacteria and yeast strains, the two negative chromosome aberration tests in mammaliain cells, the negative UDS test in mammalian cells and the 4 negative HPRT tests in mammalian cells, the genotoxicity potential of 1,2-dihydro-2,2,4-trimethylquinoline, oligomers is well examined. Not a single positive in vitro genotoxicity test is reported.

Short description of key information:
Six negative Ames tests including different bacteria and yeast strains, two negative chromosome aberration tests in mammalian cells, a negative UDS test in mammalian cells and 4 negative HPRT tests in mammalian cells, tfor TMQ ( 1,2-dihydro-2,2,4-trimethylquinoline, oligomers) are available

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information no classification is required according to the EU classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).