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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-04-18 - 2012-07-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Guideline No. 487 "In vitro Mammalian Cell Micronucleus Test".
Deviations:
yes
Remarks:
slight modification of the expression phase and harvest time, which is not compromising the outcome of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): sodium 3-mercaptopropanesulphonate
- Substance type: organic
- Physical state: yellowish powder
- Analytical purity: approx. 85 %, dose calculation adjusted to purity
- Storage condition of test material: at room temperature, moisture protected,
- Stability in solvent: stable

Method

Species / strain
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (23 years old) for the first experiment, from a 27 year-old female donor for Experiment IIA and from a 33 year-old female donor for Experiment IIB. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats
Test concentrations with justification for top dose:
Experiment I without S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment IIA without S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment I with S9-mix: 0, 13.6, 23.8, 41.7, 73.0, 127.8, 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Experiment IIA and B with S9-mix: 223.6, 391.3, 684.7, 1198.3, 2097.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water, the final concentration of deinonised water in the culture medium was 10 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent solvent controls (culture medium with 10 % deionised water) (local tap water deionised at Harlan CCR)).
Positive controls:
yes
Remarks:
Mitomycin C (2.0 µg/mL - Exp. 1), Demecolcin (150 ng/mL - Exp. 2), Cyclophosphamide (10 µg/mL - Exp. 1 and 2)
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Remarks:
Dilutions of stock solutions were prepared on the day of the experiment. Stability of Demecolcin, Mitomycin C and CPA in solution is unknown but a mutagenic response in the expected range is sufficient biological evidence of chemical stability.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40 hours
- Exposure duration: 4 and 20 hours
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: at least 1000 binuecleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: other: To describe a cytotoxic effect the CBPI (cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis (7).

OTHER:
Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data.
b) The positive control substances should produce significant increases in the number of cells with micronuclei.

Evaluation of Results

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results
Species / strain:
lymphocytes: human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In absence and presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. In exp. IIA without S9 mix stat. sig. increases (0.8, 1.0 % micronucleated cells) were in range of historical controls.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on pH value was observed.
- Effects of osmolality: No relevant influence on osmolarity value was observed.
- Precipitation: No precipitation of the test item in the culture medium was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The test item sodium 3-mercaptopropanesulphonate, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix in three independent experiments. The following study design was performed:

 

Without S9-Mix

With S9-Mix

 

Exp. I

Exp. IIA

Exp. I and IIB

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Preparation interval

40 hrs

40 hrs

40 hrs

Total culture period

88 hrs

88 hrs

88 hrs

So, in Experiment I, the exposure period was 4 hours with and without S9 mix and in Experiment IIA, the exposure period was 20 hours without S9 mix. In Experiment IIB, the exposure period was 4 hours with S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostatis.

The highest applied concentration in this test on toxicity (2097.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (85 %) of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and in accordance with OECD Guideline 487.

No precipitation of the test item in the culture medium was observed. No relevant influence on osmolarity or pH value was observed.

No relevant cytotoxicity, indicated by reduced CBPI and described as cytostasis could be observed up to the highest applied concentration.

In the absence and the presence of S9 mix, no biologically relevant increase in the number of cells carying micronuclei was observed.

The micronucleus rates of the cells after treatment with the test item (0.25 - 1.00 % micronucleated cells) were close to the range of the solvent control values (0.20 - 0.65 % micronucleated cells) and within the range of the laboratory historical control data. However, in Experiment IIA in the absence of S9 mix statistically significant increases (0.8 and 1.0 % micronucleated cells) were observed after treatment with 223.6 and 2097.0 µg/mL. The values are in the range of the historical solvent control data (0.05 - 1.45 %micronucleated cells) and therefore biologically irrelevant.

Appropriate mutagens were used as positive controls. In both experiments, either Demecolcin (150.0 ng/mL), MMC (2.0 µg/mL) or CPA (10.0 µg/mL) were used as positive controls and showed distinct statistically significant increases in cells with micronuclei.

Conclusion:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes, when tested up to the highest required concentration.

Therefore, sodium 3-mercaptopropanesulphonate is considered to benon-mutagenic in this in vitro micronucleustest, when tested up to the highest required concentration.

Table 2: Summary of results of the in vitro micronucleus test in human lymphocytes with sodium 3-mercaptopropanesulphonate

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Negative control

2.00

 

0.45

 

 

Solvent control1

1.98

 

0.25

 

 

Positive control2

1.74

25.7

11.90S

 

 

684.7

2.05

n.c.

0.45

 

 

1198.3

2.11

n.c.

0.25

 

 

2097.0

2.10

n.c.

0.35

Exposure period 20 hrs without S9 mix

IIA

40 hrs

Negative control

1.96

 

0.25

 

 

Solvent control1

1.90

 

0.25

 

 

Positive control3

1.51

46.9

3.00S

 

 

223.6

1.80

11.0

0.80S

 

 

1198.3

1.69

23.2

0.50

 

 

2097.0

1.67

25.2

1.00S

Exposure period 4 hrs with S9 mix

I

40 hrs

Negative control

1.82

 

0.30

 

 

Solvent control1

2.01

 

0.20

 

 

Positive control4

1.58

29.6

4.40S

 

 

684.7

1.92

9.7

0.30

 

 

1198.3

1.91

10.2

0.45

 

 

2097.0

1.90

11.6

0.35

IIB

40 hrs

Negative control

2.01

 

0.70

 

 

Solvent control1

1.91

 

0.65

 

 

Positive control4

1.71

29.7

6.00S

 

 

684.7

1.93

n.c.

1.00

 

 

1198.3

1.95

n.c.

0.85

 

 

2097.0

1.97

n.c.

0.65

*    For the positive control groups, the relative values are related to the negative controls; for the test item treatment groups the values are

related to the solvent controls

**     The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

1       Deionised water 10.0 % (v/v)

2           MMC                      2.0 µg/mL

3       Demecolcin     150.0 ng/mL

4           CPA                     10.0 µg/mL

Table 3: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

4 hrs

40 hrs

2.00

---

Solvent control

4 hrs

40 hrs

1.98

---

13.6

4 hrs

40 hrs

n.d.

n.d.

23.8

4 hrs

40 hrs

n.d.

n.d.

41.7

4 hrs

40 hrs

n.d.

n.d.

73.0

4 hrs

40 hrs

n.d.

n.d.

127.8

4 hrs

40 hrs

n.d.

n.d.

223.6

4 hrs

40 hrs

2.02

n.c.

391.3

4 hrs

40 hrs

2.06

n.c.

684.7

4 hrs

40 hrs

2.05

n.c.

1198.3

4 hrs

40 hrs

2.11

n.c.

2097.0

4 hrs

40 hrs

2.10

n.c.

With S9 mix

Negative control

4 hrs

40 hrs

1.82

---

Solvent control

4 hrs

40 hrs

2.01

---

13.6

4 hrs

40 hrs

n.d.

n.d.

23.8

4 hrs

40 hrs

n.d.

n.d.

41.7

4 hrs

40 hrs

n.d.

n.d.

73.0

4 hrs

40 hrs

n.d.

n.d.

127.8

4 hrs

40 hrs

n.d.

n.d.

223.6

4 hrs

40 hrs

1.94

7.0

391.3

4 hrs

40 hrs

1.87

13.9

684.7

4 hrs

40 hrs

1.92

9.7

1198.3

4 hrs

40 hrs

1.91

10.2

2097.0

4 hrs

40 hrs

1.90

11.6

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*     Mean value of two cultures
n.d. Not determined
n.c.   Not calculated as the CBPIwas equal or higher than solvent control value

Toxicity - Experiment IIA

In Experiment IIA the CBPI in two cultures (500 cells per culture) was determined.

Table 4: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

20 hrs

40 hrs

1.96

---

Solvent control

20 hrs

40 hrs

1.90

---

13.6

20 hrs

40 hrs

n.d.

n.d.

23.8

20 hrs

40 hrs

n.d.

n.d.

41.7

20 hrs

40 hrs

n.d.

n.d.

73.0

20 hrs

40 hrs

n.d.

n.d.

127.8

20 hrs

40 hrs

n.d.

n.d.

223.6

20 hrs

40 hrs

1.80

11.0

391.3

20 hrs

40 hrs

1.78

13.8

684.7

20 hrs

40 hrs

1.70

22.0

1198.3

20 hrs

40 hrs

1.69

23.2

2097.0

20 hrs

40 hrs

1.67

25.2

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*       Mean value of two cultures
n.d.   Not determined

Toxicity - Experiment IIB

In Experiment IIB the CBPI in two cultures (500 cells per culture) was determined.

Table 5: Cytotoxicity of sodium 3-mercaptopropanesulphonate to the cultures of human lymphocytes.

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

4 hrs

40 hrs

2.01

---

Solvent control

4 hrs

40 hrs

1.91

---

223.6

4 hrs

40 hrs

1.97

n.c.

391.3

4 hrs

40 hrs

1.94

n.c.

684.7

4 hrs

40 hrs

1.93

n.c.

1198.3

4 hrs

40 hrs

1.95

n.c.

2097.0

4 hrs

40 hrs

1.97

n.c.

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*     Mean value of two cultures
n.c. Not calculated as the CBPI was equal or higher than solvent control value



Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The study was performed according to the OECD Guideline 487 with deviations (expression phase and harvest time were slightly modified - these deviations are not considered to influence the outcome of the study) and is considered to be of the highest quality (reliability Klimisch 1). The vehicle water and the positive control substances fulfilled validity criteria of the test system. The positive controls mitomycin C, demecolcin and cyclophosphamide induced micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. None of the cultures treated with 3-mercaptopropanesulphonate in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of micronuclei. Based on this test, 3-mercaptopropanesulphonate is considered not to be non-mutagenic in human lymphocytes.
Executive summary:

The test item 3-mercaptopropanesulphonate, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro (Bohnenberger, 2012). Cultured human lymphcytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2a without S9 -mix (exposure period 20 hrs, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and 2b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalasin B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2097.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight and the purity (85 %) of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, in experiment IIa in the absence of S9 mix statistically significant increases (0.8 and 1.0 % micronucleated cells) were observed after treatment with 223.6 and 2097.0 µg/mL. The values are clearly within the range of the historical control data (0.05 - 1.45 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, sodium 3 -mercaptopropanesulphonate is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.