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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
12-May-2010 to 21 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Sodium thiocyanate
- Substance type: White crystalline powder
- Physical state: Solid
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark in desiccator

Method

Species / strain
Species / strain:
lymphocytes: human peripheral blood
Details on mammalian cell lines (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 10, 33, 100, 333 and 811 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 10, 33, 100, 333 and 811 µg/mL
First cytogenetic test:
Without Sand with S9-mix, 3 h exposure time, 24 h fixation time: 100, 333 and 811 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 333 and 811 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 100, 333 and 811 µg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 100, 333 and 811 µg/mL
Vehicle:
- Vehicle(s)/solvent(s) used: RPMI 1640 culture medium
- Justification for choice of solvent/vehicle: Test compound was soluble in culture medium and this has been accepted and approved by authorities and international guidelines
Controlsopen allclose all
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 811 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 811 µg/mL (= 0.01 M)

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.


Any other information on results incl. tables

No effects of Sodium thiocyanate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Sodium thiocyanate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Sodium thiocyanate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that Sodium thiocyanate is not clastogenic in human lymphocytes under the experimental conditions described in the report.
Executive summary:

Evaluation of the ability of Sodium thiocyanate to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment).

This report describes the effect of Sodium thiocyanate on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of Sodium thiocyanate was tested in two independent experiments.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 0600801070919-0 (date Jan. 09, 2008) of Sodium thiocyanate was a white crystalline powder with a purity of 99.9% (dried material). Sodium thiocyanate was dissolved in RPMI 1640 medium.

In the first cytogenetic assay, Sodium thiocyanate was tested up to 811 μg/ml (= 0.01 M) for a

3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v)

S9-fraction. This is the highest concentration recommended in the guidelines.

In the second cytogenetic assay, Sodium thiocyanate was tested up to 811 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix Sodium thiocyanate was also tested up to 811 μg/ml for a 3 h exposure time with a 48 h fixation time.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Sodium thiocyanate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

No effects of Sodium thiocyanate on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Sodium thiocyanate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that Sodium thiocyanate is not clastogenic in human lymphocytes under the experimental conditions described in this report.