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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat (Sprague-Dawley strain), male, liver homogenate - S9 mix
Test concentrations with justification for top dose:
12, 37, 111, 333, 1000 and 3000 µg/plate
Untreated negative controls:
yes
Remarks:
0 µg/plate of test material
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used with tester stratins TA100 and TA1535 at 0.5 µg/plate
Untreated negative controls:
yes
Remarks:
0 µg/plate of test material
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Used with tester strains WP2 uvrA and TA98 at 0.5 µg/plate
Untreated negative controls:
yes
Remarks:
0 µg/plate of test material
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Used with tester starins TA1537 at 50 µg/plate
Untreated negative controls:
yes
Remarks:
0 µg/plate of test material
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene
Remarks:
Used with tester strains TA100 and TA98 at 0.4 µg/plate and tester strains TA1535 and TA1537 at 2 µg/plate and tester strain WP2 uvrA at 4 µg/plate
Details on test system and experimental conditions:
Description of follow up repeated study: Preliminary test had carried out to decide the appropriate starting dose level of the main study at the concentration of 1.6, 8, 40, 200, 1,000 and 3,000 μg/plate. Criteria for evaluating results: the number of revertant colonies in the plate was counted after 2 days incubation at 37 °C.

The direct incorporation method: For test without metabolic activation, the test substance and 0.1 ml of fresh bacterial culture were added to 2.0 ml of overlay agar. For tests with metabolic activation, 0.5 ml of metabolic activation mixture containing an adequate amount of postmitochondrial fraction was added to the overlay agar after the addition of the bacteria and test substance. All plates in a given test should be incubated for the same time period.





Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
not valid

Table 1: Result of bacterial reverse mutation assay with calcium sulfate, dihydrate.

Tester strain

Chemical treated

Dose (µg/plate)

Colonies/plate (mean)[Factor]

Without S-9 mix

With S-9 mix

TA100

Test item

0

12

37

111

333

1,000

3,000

132

139 [1.0]

143 [1.1]

125 [0.9]

129 [1.0]

124 [0.9]

124 [0.9]

114

115 [1.0]

122 [1.1]

115 [1.0]

125 [1.0]

106 [1.0]

115 [1.0]

TA 1535

Test item

0

12

37

111

333

1,000

3,000

13

20 [1.5]

22 [1.7]

17 [1.3]

16 [1.2]

17 [1.3]

14 [1.1]

10

11 [1.1]

12 [1.2]

13 [1.3]

14 [1.4]

13 [1.3]

9 [0.9]

TA 1537

Test item

0

12

37

111

333

1,000

3,000

15

16 [1.0]

11 [0.7]

15 [1.0]

14 [0.9]

13 [0.9]

12 [0.8]

17

17 [1.0]

22 [1.3]

19 [1.1]

17 [1.0]

16 [0.9]

16 [0.9]

E.coli

WP2uvrA

Test item

0

12

37

111

333

1,000

3,000

8

7 [0.9]

6 [0.8]

8 [1.0]

8 [1.0]

6 [0.8]

6 [0.8]

 

12

8 [0.7]

13 [1.1]

8 [0.7]

8 [0.7]

9 [0.8]

9 [0.8]

Positive controls

 

 

 

TA100

TA1535

TA98

TA1537

WP2uvrA

TA100

TA1535

TA98

TA1537

WP2uvrA

SA

SA

4NQO

9-AA

4NQO

2-AA

2-AA

2-AA

2-AA

2-AA

0.5

0.5

0.5

50

0.5

0.4

2

0.4

2

4

493 [3.7]

371 [28.5]

426 [19.4]

740 [49.3]

377 [47.1]

 

 

 

 

14 [0.6]

 

 

 

 

 

491 [4.3]

394 [39.4]

289 [8.5]

326 [19.2]

311 [25.9]

[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: 2-aminoanthracene

Conclusions:
Interpretation of results: negative With and without metabolic activation

Mutation in the Salmonella tryphimurium (strains TA 98, TA100, TA 1535 and TA 1537) and in the Escherichia coli WP2 uvrA did not occur with calcium sulfate, dihydrate
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-13 to 2010-01-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and Guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
In accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK +/- locus (thymidine kinase heterozygote system)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Prior to freezing stocks of the cells, they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Concentrations were as calcium sulfate
Preliminary toxicity test: 0, 5.32, 10.63, 21.27, 42.53, 85.06, 170.13, 340.25, 680.5 and 1361 µg/mL in the presence and absence of S9

Experiment 1: 21.25, 42.5, 85, 170, 340, 453.33, 566.66 and 680 µg/mL in the absence of S9. 85.06, 170.13, 340.25, 680.5, 1020.75 and 1361 µg/mL in the presence of S9.

Experiment 2: 25, 50, 100, 150, 200, 250, 300 and 350 µg/mL in the absence of S9. 85.06, 170.13, 340.25, 680.5, 1020.75 and 1361 µg/mL in the presence of S9.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: R0 medium
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: (EMS) in the absence of metabolic activation at 400 µg/mL in Experiment 1 and 150 µg/mL in Experiment 2.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: (CP) 2 µg/mL in the presence of metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: in Experiment 1, cells were treated with the test material for 4 hours both in the presence and absence on metabolic activation. In Experiment 2, cells were treated with the test material for 4 hours in the presence of metabolic activation and 24 hours in the absence of metabolic activation.
- Expression time : 2 days
- Selection time : up to 48 hours

SELECTION AGENT :5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: performed in duplicate (denoted as A and B)

NUMBER OF CELLS EVALUATED: 10^4 cells/mL (2000 cells/well) plated in selective medium for evaluation of mutant frequency and 10 cells/mL (2 cells/well) to assess viability (%V) plated in non-selective medium.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER: MTT (2.5 mg/mL in PBS) was added to each well of the mutation plates to aid in counting viable cells.
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should be within this range, minor errors may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and are repeated.
Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10-6 will be considered positive. If a test material produces a modest increase in mutant frequency, only marginally exceeding the GEF and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. However when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.
Small significant increases designated by the UKEMS statistical package were reviewed using the above criteria (and where necessary disregarded at the Study Director's discretion).
Statistics:
The experimental data was analysed using a dedicated computer program which follows the statistical guidelines recommended by the UKEMS.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
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 experiment.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
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. The 24-hour exposure without metabolic activation demonstrated that th
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked change was noted in the pH when the test material was added to the media.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm when test material was dosed into the media
- Precipitation: In the preliminary toxicity test, a precipitate of the test material was observed at and above 170.13 µg/mL in all three of the exposure groups. In Experiment 1 a precipitate of the test material 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.

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data were included. All controls performed within the study were found to be valid.

RANGE-FINDING/SCREENING STUDIES: Please refer to Table 1 in the attached Appendix 1 Tabulated data for full results of the range-finding study. At 4 and 24-hours in the absence of metabolic activation there were marked dose-related reductions in the Relative Suspension Growth (%RSG) of treated cells. A slight reduction in %RSG occurred at the 10 mM limit dose in with S9. The toxicity curve was very steep in both the 4-hour and 24-hour exposure groups in the absence of metabolic activation. A precipitate was observed at concentrations ≥ 170.13 µg/mL in all three groups. In the mutagenicity experiments the maximum dose for the 4 and 24-hour exposure groups without S9 was limited by toxicity. The maximum dose level for the 4-hour exposure group in the presence of metabolic activation was the 10 mM limit dose.

For tabulated data on results, please refer to Appendix 1 Tabulated data for Flanders 2010.

Controls

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.

Toxicity

In 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.

In 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 material.

The author 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.

Experiment 1

The test 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.

Experiment 2

The test 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

Conclusions:
Interpretation of results: negative in the presence and absence of metabolic activation

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

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 experiment.

Conclusion. The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.


Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

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

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed to GLP and guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 weeks
Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:Calcium sulfate, dihydrate was dissolved in 1 % CMC (Sodium carboxymethyl Cellulose) and supersonic wave was used to prepare the highest dose concentration.



Duration of treatment / exposure:
1 day
Frequency of treatment:
Single treatment
Dose / conc.:
1 250 mg/kg bw/day
Dose / conc.:
2 500 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
6
Positive control(s):
- mitomycin C
- Doses / concentrations: 0.5 mg/kg and 1.0 mg/kg
Details of tissue and slide preparation:
To observe the cell multiplication of bone marrow, a specimen was fixed with methanol. And 4 % Giemsa solution was used for dyeing to observe the ratio of polychromatic erythrocytes. To observe the micronucleus in the polychromatic erythrocytes, 40 μg/ml acridine was dropped for dyeing



Evaluation criteria:
At least 2000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei
Statistics:
ANOVA (using Sigmastat 2.0 statistic programme)
Sex:
male
Genotoxicity:
negative
Remarks on result:
other: Calcium sulfate dihydrate showed negative results in the micronucleus test in vivo up to the test concentration of 5000 mg/kg b.w

Table 1: Effect on mitotic index or PCE/NCE ratio by dose level.

Dose (mg/kg)

Group mean

(PCE/(PCE+NCE)) (%)

Group mean frequency of

MNPCE

(per 1,000)

Vehicle

1,250

2,500

5,000

Positive control (0.5mg/kg)

Positive control (1.0mg/kg)

60.49±7.77

56.58±9.07

54.61±6.13

54.48±9.02

46.58±10.76

46.96±7.08

6.5

7.0

7.7

8.0

19.2

44.8

Conclusions:
negative
Calcium sulfate dihydrate showed negative results in the micronucleus test in vivo up to the test concentration of 5000 mg/kg b.w
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

It is concluded that the available data indicate that calcium sulfate has no genotoxicity and therefore does not warrant classification for mutagenicity under CLP.