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EC number: 232-092-5
CAS number: 7786-17-6
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,
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.
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
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
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
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.
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,
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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