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EC number: 202-853-6
CAS number: 100-44-7
In vitro bacterial
The in vitro mutagenicity of benzyl
chloride has been tested in in vitro bacterial systems mainly
based on the Ames test. The three reported studies are all similar to
the OECD guideline 471.
In a study from Brusick (1976), the authors
tested the mutagenicity and recombinogenicity of benzylchloride (CAS n°
100-44-7) in the absence and presence of a metabolizing system with a
methodology similar to the OECD guideline 471. The Salmonella
typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and
the Saccharomyces cerevisiae strain D4 were used and the
metabolizing system was obtained from rat liver homogenates. These
strains were exposed to 0.1, 1, 5 and 10 µL/plate with and without
metabolic activation for 48 to 72 h. Solvent controls and positive
controls were performed.
The test showed that benzylchloride was
mutagenic for Salmonella typhimurium strain TA 100 and
recombinogenic for Saccharomyces cerevisiae strain D4. This
respons was observed in absence and presence of the metabolic activation
system, demonstrating that metabolic activation is not essential for
mutagenicity or recombinogenicity. Furthermore, there was also an
indication of a weak response at the high dose of the Salmonella
typhimurium strain TA 1535. Effects were only seen at relatively
high doses and dose-related responses at the lower end of the dose range
were not observed. Hence, based on this information, further in vitro testing
would be required with other systems to clarify if in vivo testing
would be necessary.
In an other study from Flowers (1976), the
authors tested the mutagenicity of benzylchloride (CAS n° 100-44-7) in
the absence and presence of a mammalian metabolizing system with a
methodology similar to the OECD guideline 471.Salmonella typhimurium strains
TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. These strains
were exposed to 0.001, 0.01, 0.1, 1, 5 and 10 µL/plate of the test
substance (in DMSO) with and without mammalian metabolic activation.
Solvent control and positive controls were performed. However, the rules
for the interpretation of the results are not reported.
Hence, the authors concluded that
benzylchloride was mutagenic for Salmonella typhimurium strain TA
100 at two dose levels (5 and 10 µL/plate) with metabolic activation.
Questionable results were besides observed for strains TA 98 and TA 1538
at 5 µL/plate without metabolizing system. For all strains except TA
100, the authors noticed toxic effects at the highest tested
concentration, 10 µL/plate.
Considering, the criteria in the OECD 471
for the interpretation of mutagenicity tests, the results should be
interpretated as ambiguous since no clear dose-response relation was
established. However, the overall results proves that benzylchloride
shows obvious signs of mutagenicity in the tested Salmonella strains.
Hence, further in vitro testing would be required to clarify the
Finally, in an other study from
Hyldig-Nielsen (1981), the authors tested the mutagenicity potential of
benzyl chloride (CAS n° 100 -44 -7) in a Salmonella spot test according
to the methodology of Ames et al. (1975). They imbibed discs of the test
substance diluted in ethanol at different concentrations and applied the
discs in petri dishes innoculated with the S. TA strains. They conducted
two set of experiments. The first set included five common strains of
Salmonella (TA 1535, TA 1537, TA 1538, TA 98, TA 100) and four
concentrations as a solvent control and as
positive control with 2 -Amino-anthracene,
tested all with and without metabolic activation. The second experiment
was conducted with three TA strains (TA 1538, TA 98, TA 100) and five
different concentrations, again with and without metabolic activation.
Results of the first sub-set of experiment
are not reported as the applications of several discs per petry dish
disturbed and avoided a clear reading of the results. However in the
second experiment (with one disc per petry dish), they authors found a
high increase of revertants in the highest tested concentration with and
without metabolic activation.
They concluded that benzyl chloride is a
potent mutagen in the Salmonella spot test. However, no explanation is
given on their referential for interpretating these results. According
to the OECD guideline 471, criteria to conclude on positive mutagenicity
in in vitro bacterial reverse mutation assays are complex and
varied but nonetheless, a significant increase in the number of
revertants and a clear dose-response relation ship should established.
At the present level of information, we may hypothesize that the
increase in the number of revertants was their criteria. However, signs
of toxicity were observed at the highest tested concentrations.
Therefore, these results should be interpretated with caution and
regarded as ambiguous. Hence, further in vitro testing would be
required to clarify the situation.
The results of these experiments are
similarly described and all based on the Ames test method. The
differences between the Brusick study and the Flowers study, which are
the more relevant and reliable, are not really big. Both exhibit
deviations to the guideline. However, in the Flowers study (1976), no
data is reported on the interpretation criteria of the mutagenicity and
based on the OECD criteria, these results would be ambiguous. Thus,
these elements were the basis for the choice of the Brusick study (1976)
as a key study.
Altogether, these results bring evidences on
the mutagenicity potential of benzyl chloride in in vitro bacterial
system. Hence based on all available information, benzyl chloride should
be considered as a mutagen in in vitro bacterial system.
So far, based on all results, further
testing as required in the annex VIII should be required. However,
according to the column 2 of the specific rules for adaptation from
column 1 of the REACH regulation n° 1907/2006, an in vitro study
on cytogenicity in mammalian cells or an in vitro micronucleus
study of the annex VIII requirement does not usually need to be
conducted if the substance is known to be carcinogenic category 1 or 2
or mutagenic category 1, 2 or 3. In this case, benzyl chloride is
classified in the CLP regulation n° 1272/2008 EC as a carcinogen
category 1B, hence no study on cytogenicity in mammalian cells or an in
vitro micronucleus is required.
In vivo Mammalian
The in vivo mutagenicity of benzyl
chloride has been tested in in vivo Mammalians systems mainly
based on the in vivo micronucleus test. Two reported studies are
similar to the OECD guideline 474 and one test is based on the sperm
head abnormality test.
In a study from Sison (1990), the authors
tested the mutagenicity of benzyl chloride in vivo according to a
methodology similar to the OECD guideline 474 (Mammalian Erythrocyte
Micronucleus Test) on Swiss-Webster male and female mice. Eight mice per
dose were injected intraperitoneally less than 8 mL/kg bw of benzyl
chloride solutions freshly diluted in DMSO twice. The concentrations
corresponded to 1/8 LD50, 1/4 LD50 and 1/2 LD50 previously established
in a screening test. The injections of benzyl chloride solutions took
place 30 and 6 hours prior the preparation of the bone marrow. At the
end of the exposure period, mice were sacrificed and bone marrow fluids
were collected, stained and the number of micronucleated polychromatic
erythrocytes on per thousand were counted to establish cytogenetic
damages. Results for the benzyl chloride were then compared with other
In the test conditions, the authors
established that benzyl chloride induced significantly micronucleated
polychromatic erythrocytes compared with solvent control. However, this
increase was not estimated dose dependant and the induction rate was
lower than the 3-fold threshold generally recommended. Benzyl chloride
should therefore be considered as an ambiguous in vivo potential
mutagen and would require further testing to establish its mutagenicity
on somatic cells.
In an other study from Danford (1982), the
authors tested the mutagenicity of benzyl chloride also in vivo
in a micronucleus test following a methodology similar to the OECD
guideline 474. Three dose were tested, 75, 150 and 300 mg.kg bw
corresponding to 1/8 LD50, 1/4 LD50 and 1/2 LD50 established in
screening test prior this experiment. Four mice per dose were injected
either, one dose and killed 24 hours later, or one dose and a second
identical dose 24 hours later then sacrificed 6 hours later. In each
experiment, the bone marrow was extracted from the femurs, stained and
scored for micronuclei in one thousand mature polychromatic
No increase in micronuclei in polychromatic
erythrocytes was observed at any dose. Therefore, benzyl chloride was
not be considered as a potent mutagen in vivo in Tuck To mice.
In an other study from Scott (1982), the
authors tested the mutagenicity of benzyl chloride (CAS n° 100 -44 -7)
in a sperm head abnormality test on (CBAxBALB/C) F1 male mice.Three
similar experiments were conducted. Each time, mice received five equal
consecutive doses of benzyl chloride formulated in 0.5 % Tween 80,
corresponding in one experiment to 125, 250 and 500 mg/kg bw/day
subcutaneanously and in two other experiments to 50, 100, 200 and 400
mg/kg bw/day intraperitoneally. Thirty five days later, all surviving
mice were killed and scored for sperm head abnormilties.
Considering that the criteria for a positive
response are that the increase in abnormal sperm heads shall be at least
double the negative control and be significant at least at the P>0.05
and besides, that reproducibility and a dose-response are required to
confirm a positive response, benzyl chloride did not induce a positive
response in the sperm head abnormality test.
Therefore, at this level of information,
benzyl chloride was not considered as a potent mutagen for germ cells of
mice. However as the specific target genes of this test are unknown,
this study gives only support to the other results.
All three studies are sufficiently
documented and gives relatively consistent results. The studies based on
the micronucleus test are the most relevant thanks to their recognized
testing method. Besides, altogehter these results prove that benzyl
chloride does not induce significant genetic damages in vivo. At
this level of information, further testing in vivo mutagenicity
on somatic cells does not seem to be required and either recommanded.
Short description of key information:
- In vitro bacterial mutagenicity: positive
- In vivo Mammalian mutagenicity: negative
Endpoint Conclusion: No adverse effect observed (negative)
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