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Key value for chemical safety assessment

Additional information

The potential for the registered substance to cause genotoxicity has been assessed using two bacterial gene mutation studies, a mammalian cell mutation study. Several studies, performed on several structural analogues, are provided on the basis of read-across. Read-across to these substances is considered valid due to the structural similarities between the analogues and the target substance, 2,2'-Methylenebis(6-nonyl-p-cresol).

 

IN VITRO BACTERIAL GENE MUTATION

Miltenburger (1984) determined the mutagenic potential of the registered substance in an Ames test performed following a protocol similar to the OECD 471 guideline and under GLP conditions. Accordingly the study was assigned a reliability score of 2 in line with the principles for assessing data quality as defined by Klimisch (1997). S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were exposed to the test material using the plate incorporation method at concentrations of 0, 0.1, 0.5, 1.0, 5.0 and 10.0 µL/plate. Each concentration was assayed in triplicate with and without metabolic activation (S9-mix). Exposure to the test material did not induce higher revertant rates, in the following strains; TA 1537, TA 1538, TA 98 and TA1535. Furthermore, TA 100 did not show any indication of enhanced revertant rates. Based on the relevance of enhancement factors the test material is classified as non-mutagenic, under the conditions of this test.

 

Banduhn (1985) investigated the mutagenic potential of the target substance using S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 in an Ames test, performed under GLP conditions and in accordance with the OECD guideline 471 and EU Method B.14. The study was therefore assigned a reliability score of 1 in accordance with Klimish (1997). The test material was investigated with and without metabolic activation at concentrations of 0.1, 0.5, 1.0, 5.0 and 10.0 µL/plate both (S9-mix), each dose level being assessed in triplicate. The test material caused toxic effects at concentrations starting from 0.5 µL/plate in the absence of S-9 mix and at 10.0 µL/plate in the presence of S-9 mix. Precipitation in the agar occurred starting at 1 mL/plate or higher, this did not affect colony counting. Up to the highest dose investigated, no relevant increase of revertant colony numbers was obtained with strains TA98, TA100 and TA1537 with and without metabolic activation, when compared to the corresponding controls. In the strain TA1538 a doubling in the rate of revertants was observed with metabolic activation at a concentration of 1.0 µL/plate only. Without the activating system, the number of revertants was reduced at a concentration of 1.0 µL/plate and higher when compared with the corresponding control. In the strain TA1535 enhanced rates of revertants were obtained only in the absence of activation. Compared with the control the rates were elevated at all concentrations and doubled at 1.0 and 10.0 µL/plate, respectively. With the activating system, the number of revertants was within the range of the control. Based on these observations, a reproducible increase of revertants up to a doubling was found in the strain TA1535 without metabolic activation. It was therefore be concluded that the test material showed weak mutagenic potential, inducing point mutations by base-pair changes in the genome of S. typhimurium TA1535.

 

When the findings of the studies were considered together, the test materuial was concluded not to be mutagenic to S. typhimurium.

 

IN VITRO MAMMALIAN GENE MUTATION

Verspeek-Rip (2013) determined the genotoxic potential of the registered substance in an in vitro gene mutation study using L5178Y mouse lymphoma cells. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 476, EU Method B.17 and IWGT recommendations. The study was assigned a reliability score of 1 in line with Klimisch (1997). Based on the findings of a dose range finding test the definitive study was performed as two independent experiments within the dose range 0.03 to 100 µg/mL. In experiment 1, cultures were exposed to the test material for 3 hours, with and without metabolic activation (8 % v/v S9-mix). In experiment 2, cultures were exposed for 3 hours without S9-mix, and for 24 hours with 12 % v/v S9-mix. The test material precipitated in the exposure medium at concentrations of 33 μg/mL and above. It was tested beyond the limit of the solubility to obtain adequate cytotoxicity and mutagenicity data.

The numbers of small and large colonies in treated cultures were comparable to the numbers in the solvent controls. Both in the presence and absence of metabolic activation (S9-mix), the test material did not induce a significant increase in the mutation frequency in the first experiment, results that were confirmed by the second experiment.

 

Therefore under the conditions of the study, the test material was determined to be non-mutagenic.

 

READ-ACROSS, 6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol (CAS No. 85-60-9)

Brusick (1976a) determined the genotoxic potential of the test material in an in vitro bacterial gene mutation assay (Ames test) conducted broadly in accordance with OECD guideline 471. The study was performed to sound scientific principles and reported with a sufficient level to be assigned a reliability score of 2 in line with Klimisch (1997). S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 and S. cerevisiae D4 were exposed to the test material at concentrations ranging from 0.1 to 500 µg/plate using the overlay method. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats. The test material did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

Blazak (1987) determined the genotoxic potential of the test material in an in vitro mammalian gene mutation assay. Chromosomal aberrations were investigated in a Chinese hamster ovary (CHO) cells assay performed broadly in accordance with OECD guideline 473. The study was conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Accordingly the study was assigned a reliability score of 2 in line with Klimisch (1997). CHO cells were exposed to the test material at concentrations ranging from 5 to 200 µg/mL in both the presence and absence of S-9 metabolic activation (derived from Aroclor 1254 induced rats). The cells were evaluated microscopically for mitotic indices and for chromosomal aberrations. Solvent and positive controls were included. In both the absence and presence of metabolic activation, statistical analysis revealed no significant differences among the solvent control and treated cells in the percentage of structurally aberrant cells or in the frequency of structural aberrations per cell. Under the conditions of the study, the test material did not induce chromosomal aberrations in the presence or in the absence of metabolic activation and was therefore considered to be non-clastogenic.

Mirsalis (1987) determined genotoxic potential in an in vitro DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells, conducted broadly in accordance with OECD guideline 482. The study was conducted to GLP in accordance with generally accepted scientific principles, and was assigned a reliability score of 2 in accordance with Klimisch (1997). Unscheduled DNA synthesis (UDS) tests with primary cultures of rat hepatocytes were carried out at concentrations ranging from 1 to 250 µg/mL. An increase in UDS above that of the solvent control was not observed after treatment at any of the tested concentration. These results indicated that the test material was not a genotoxic agent.

 

Based on the available in vitro data, the structural analogue, 6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol, is not expected to cause genotoxicity.

 

READ-ACROSS, 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol (CAS No. 96-69-5)

Two in vitro bacterial gene mutation assays have been provided, Brusick (1976b) and Brusick (1976c). Both studies were performed broadly in accordance with OECD 471 and followed the same study protocol. The studies pre-date GLP and the standard guidelines; nevertheless, the data are well reported and the experiments were conducted in accordance with generally accepted scientific principles. Accordingly both studies were assigned a reliability score of 2 in line with Klimisch (1997).

The test material was examined for mutagenic activity in a series of in vitro microbial assays employing S. typhimurium strains TA1535, TA 1537, TA1538, TA98 and TA 100, and S. cerevisiae strain D4 as indicator organisms. The compound was tested directly and in the presence of liver microsomal enzyme preparations from Aroclor-induced rats at concentrations between 0.1 and 500 µg/mL. The test material did not demonstrate mutagenic activity in either assay and was considered not mutagenic under the conditions of this test.

San Sebastien (1988) determined the genotoxic potential in an in vivo bone marrow cytogenetics study, performed under GLP conditions following a study protocol similar to OECD 475. The study was performed to sound scientific principles reported with a sufficient level of detail to assess the quality of the submitted data. The study was therefore assigned a reliability score of 2 in accordance with Klimisch (1997). Animals were dosed at 1400 and 700 mg/kg, based on the results of a preliminary toxicity test. Test solutions and the vehicle control (corn oil) were administered in single oral doses to three groups per dose, each consisting of two male and three female Fisher 344 rats. Animals were sacrificed at 6, 18 and 30 hours after dosing, metaphase was arrested 2 hours after dosing with 4 mg/kg bw colchicines. At sacrificed both femurs were removed from each animal and metaphase slides prepared. Slides were stained, coded and scored for chromosomal aberrations.

All rats dosed at 700 mg/kg and 1400 mg/kg exhibited mild to severe pharmacotoxic signs at all sacrifice time intervals evaluated. Statistical analysis of the data indicated that the test material did not produce statistically significant increases in the number of aberrations or in the number of aberrant metaphases at any of the three sacrifice times evaluated. Based on these results the test material was considered to be non-clastogenic in vivo.

 

Based on the available in vitro and in vivo data, the structural analogue, 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol, is not expected to cause genotoxicity.

 

OVERALL CONCLUSION

The test material is not expected to have mutagenic potential based on the in vitro evidence provided on the registered substance and the in vitro and in vivo data provided on structural analogues. Further testing of the registered substance is, therefore, considered unnecessary.


Justification for selection of genetic toxicity endpoint
A single study could not be selected, the studies presented address different types of gene mutation and are all necessary in addressing the mutagenic potential of the test material.

Short description of key information:
Bacterial Gene Mutation: Negative.
Mammalian Gene Mutation: Negative.
Supporting data on structural analogues: Negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, this substance does not meet the criteria for classification for genetic toxicity.