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EC number: 231-900-3
CAS number: 7778-18-9
In vitro gene mutation study in bacteria:
In a reliable OECD guideline study (NIER 2001) calcium sulfate dihydrate
was tested in a bacterial reverse mutation assay inSalmonella
typhimurium(strains TA 98, TA 100, TA 1535 and TA 1537) and
Escherichia coli WP2 uvrA with and without metabolic activation (S9).
The concentrations tested were12, 37, 111, 333, 1,000 and 3,000
μg/plate. No mutations occurred.
In vitro gene mutation study in mammalian cells:
In a reliable OECD guideline study (Flanders 2010) calcium sulfate
dihydrate was tested for its abilty to induce mutations in mouse
lymphoma L5178Y cells in the presence and absence of metabolic
activation. Calcium sulfate dihydrate did not induce any toxicologically
significant increases in the mutant frequency at the TK +/- locus in
L5178Y cells and wais therefore considered to be non mutagenic under the
conditions of the test.
Table 1: Result of bacterial reverse
mutation assay with calcium sulfate, dihydrate.
Without S-9 mix
With S-9 mix
[Factor]: No. of colonies of treated
plate/No. of colonies of negative control plate
SA: Sodium azide 9-AA: 9-Amino acridine
4NQQ: 4-nitroquinoline-1-oxide 2-AA:
For tabulated data on
results, please refer to Appendix 1 Tabulated data for Flanders 2010.
The positive controls
produced marked increases in the mutant frequency per viable cell
indicating that the test system was operating satisfactorily and that
the metabolic activation system was functional. Vehicle control mutant
frequency values were within the acceptable range of 50 to 200 x 10-6viable
cells. Both positive controls induced acceptable levels of toxicity.
Experiment 1, evidence of toxicity following exposure to the test
material in the absence of metabolic activation was noted. The
modest reduction observed in the presence of metabolic activation in the
preliminary toxicity test was not reproduced. No
significant reductions in viability (%V) in either the absence or
presence of metabolic activation occurred indicating no residual
toxicity. Near optimum levels of toxicity were achieved in the absence
of metabolic activation. Optimum
levels of toxicity were not achieved in the absence of metabolic
activation due to the sharp onset of toxicity (despite a very narrow
dose interval). However,
a dose level that exceeded the usual acceptable upper limit of toxicity
was plated for viability andresistance.
Experiment 2, a marked dose-related reduction occurred in % and RTG
values in cultures dosed with the test material in the absence of
metabolic activation and no evidence of any reductions in the presence
of metabolic activation. There were no significant reductions in
viability (%V) with or without metabolic activation, indicating no
residual toxicity. Optimum
levels of test material‑induced toxicity were achieved in the absence of
metabolic activation. The
24-hour exposure without metabolic activation demonstrated that the
extended time point had a marked effect on the toxicity of the test
concluded that with no evidence of any toxicologically significant
increases in mutant frequency at any of the dose levels, including the
dose level that exceeded the usual upper limit of acceptable toxicity or
in the 24-hour exposure group of Experiment 2 where optimum levels of
toxicity were achieved, the test material had been adequately tested.
material did not induce any statistically significant or dose related
(linear-trend) increases in the mutant frequency x 10-6 per
viable cell in the absence of metabolic activation. In
the presence of metabolic activation, a very modest dose related (linear
trend) statistically significant response was observed. However,
statistically significant increases in mutant frequency were not
observed at any of the of the individual dose levels, thewas not
exceeded at any of the individual dose levels, the mutant frequency
values observed at 1020 and 1361 µg/mL only marginally exceeded the
upper acceptable range for vehicle controls, and the response was not
reproduced in Experiment 2. Therefore,
the response was considered to be of no toxicological significance. A
precipitate of the test material was observed at ≥85 µg/mL in the
absence of metabolic activation, and ≥340.25 µg/mL in the presence of
metabolic activation, this was considered not to affect the purpose and
integrity of the study.
material did not induce any statistically significant or dose related
(linear-trend) increases in the mutant frequency x 10-6per
viable cell in either the absence or presence of metabolic activation.
The test material precipitated at ≥100 µg/mL without S9, and at
≥170.13 µg/mL with S9
Introduction. The study was conducted
according to a method that was designed to assess the potential
mutagenicity of the test material on the thymidine kinase, TK +/-, locus
of the L5178Y mouse lymphoma cell line. The method
used meets the requirements of the OECD (476) and Method B17 of
Commission Regulation (EC) No. 440/2008 of.
Methods. Two independent experiments
were performed. In Experiment 1, L5178Y TK +/- 3.7.2c
mouse lymphoma cells (heterozygous at the thymidine kinase locus) were
treated with the test material at up to eight dose levels, in duplicate,
together with vehicle (solvent) and positive controls using 4-hour
exposure groups both in the absence and presence of metabolic activation
(2% S9). In Experiment 2, the cells were treated with
the test material at up to eight dose levels using a 4‑hour exposure
group in the presence of metabolic activation (1% S9) and a 24‑hour
exposure group in the absence of metabolic activation.
The dose range of test material was selected following the results of a
preliminary toxicity test. The dose range for
Experiment 1 was 21.25 to 680 µg/ml in the absence of metabolic
activation and 85.06 to 1361 µg/ml in the presence of metabolic
activation. The dose range for Experiment 2 was 25 to
350 µg/ml in the absence of metabolic activation, and 85.06 to 1361
µg/ml in the presence of metabolic activation.
Results. The maximum dose level used was
the 10 mM limit dose in the presence of metabolic activation, and was
limited by test material induced toxicity in the absence of metabolic
activation. In Experiment 1 a precipitate of the test
material was observed at and above 85 µg/ml in the absence of metabolic
activation, and at and above 340.25 µg/ml in the presence of metabolic
activation. In Experiment 2 a precipitate of the test
material was observed at and above 100 µg/ml in the absence of metabolic
activation, and at and above 170.13 µg/ml in the presence of metabolic
activation. The vehicle (solvent) controls had
acceptable mutant frequency values that were within the normal range for
the L5178Y cell line at the TK +/- locus. The positive
control materials induced marked increases in the mutant frequency
indicating the satisfactory performance of the test and of the activity
of the metabolising system.
The test material did not induce any toxicologically significant
dose-related increases in the mutant frequency at any dose level, either
with or without metabolic activation, in either the first or the second
Conclusion. The test material was
considered to be non-mutagenic to L5178Y cells under the conditions of
In vivo micronucleus assay:
In a reliable OECD guideline study (NIER 2002) male mice were
given 1,250, 2,500 and 5,000 mg/kg bw doses of calcium sulfate
dihydrate. Bone marrow was sampled 24h after the last dose and PCE/NCE
ratio determined.Calcium sulfate dihydrate showed negative
results in the micronucleus test in vivo up to the test concentration of
5000 mg/kg bw
Table 1: Effect on mitotic index or PCE/NCE
ratio by dose level.
Group mean frequency of
Positive control (0.5mg/kg)
Positive control (1.0mg/kg)
It is concluded that the available data indicate that calcium
sulfate has no genotoxicity and therefore does not warrant
classification for mutagenicity under CLP.
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