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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation including read-across data, there is no evidence for genotoxic properties of Guanidine Thiocyanate.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-02 to 2015-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch 1507511210
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction (phenobarbital and ß-naphthoflavone induced)
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 2500, and 5000 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 2- aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: experiment I in agar (plate incorporation); experiment II preincubation

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: at least 48 h at 37 °C

NUMBER OF REPLICATIONS: 3 plates per concentration and strain

DETERMINATION OF CYTOTOXICITY
- Method: background lawn, reduction in the number of revertants down to a mutation factor of approx. < 0.5 in relation to the solvent control.

OTHER: Colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH).
Tester strains TA 1535 and TA 1537 were counted manually.
Evaluation criteria:
Validity:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin
- the negative control plates with and without S9 mix are within laboratory historical ranges
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity:
A test item is considered as mutagenic if:
- a clear dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.

Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Conclusions:
Guanidine Thiocyanate is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium were exposed to Guanidinium Thiocyanate, at concentrations of 31.6, 100, 316, 1000, 2500, and 5000 µg/plate in two independent experiments. The first experiment was conducted as plate incorporation assay, the second as pre-incubation test, both in the absence and presence of a mammalian metabolic activation.

No precipitation of the test item was observed in any of the experiments. In experiment I no toxic effects were noted up to the highest concentration. In experiment II in some strains toxic effects were observed at 2500 and 5000 µg/plate.

No biological relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Guanidine Thiocyanate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagen induced a distinct increase of revertant colonies indicating the validity of the experiments.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Generally it is assumed that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate. Therefore both ions, independently of their source, can be considered relevant for assessment of genetic toxicity potential.
Thus theoretically salts of both ions with a relatively nontoxic counter ion are suitable to assess the genetic toxicity potential of Guanidinium Thiocyanate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to attached document.

3. ANALOGUE APPROACH JUSTIFICATION
Refer to attached document.

4. DATA MATRIX
Refer to attached document.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
other: clonal sub-line of a Chinese hamster fibroblast cell line (CHL)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes:
Remarks:
human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: data from Guanidine nitrate
Conclusions:
Built on the hypothesis that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate, in vitro cytogenicity was assessed based on data from in-vitro chromosome aberration studies according to OECD Guideline 473 with Sodium Thiocyante in cultured peripheral human lymphocytes and Guanidine Nitrate in Chinese hamster fibroblast cell line (CHL).

From both studies there is no evidence of induction of chromosome aberrations. In conclusion Guanidinium Thiocyanate is considered to be not clastogenic in mammalian cells.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Generally it is assumed that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate. Therefore both ions, independently of their source, can be considered relevant for assessment of genetic toxicity potential.
Thus theoretically salts of both ions with a relatively nontoxic counter ion are suitable to assess the genetic toxicity potential of Guanidinium Thiocyanate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to attached document.

3. ANALOGUE APPROACH JUSTIFICATION
Refer to attached document.

4. DATA MATRIX
Refer to attached document.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 600µg/mL and above without S9 mix following 24 hours exposure.
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: obtained from Sodium Thiocyanate study
Conclusions:
Built on the hypothesis that Guanidinium Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate, in-vitro gene mutation in mammalian cells was assessed based on data from mammalian gene mutation assays according to OECD Guideline 476 with Guanidine Nitrate and Sodium Thiocyante in L5178 Y (mouse lympoma thymidine kinase locus) cells.
From both studies there is no evidence of induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

In conclusion Guanidinium Thiocyanate is considered to be non-mutagenic in mammalian cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In-vitro studies

Ames Test

Guanidine Thiocyanate

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium were exposed to Guanidine Thiocyanate, at concentrations of 31.6, 100, 316, 1000, 2500, and 5000 µg/plate in two independent experiments. The first experiment was conducted as plate incorporation assay, the second as pre-incubation test, both in the absence and presence of a mammalian metabolic activation.

No precipitation of the test item was observed in any of the experiments. In experiment I no toxic effects were noted up to the highest concentration. In experiment II in some strains toxic effects were observed at 2500 and 5000 µg/plate.

 

No biological relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Guanidine Thiocyanate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagen induced a distinct increase of revertant colonies indicating the validity of the experiments.

Guanidine Nitrate

Additional information is available from the read-across substance Guanidine Nitrate from a reverse gene mutation assay in bacteria according to OECD guideline 471, 1983 in tester strains TA 98, TA 100TA 1535, TA l537 and TA l538 of S. typhimurium.

Guanidine Nitrate was tested up the limit concentration of 5000 µg/plate. No cytotoxicity and no increase in the number of revertants were observed in all tester strains, with or without metabolic activation.

Data were included in the data set to sustain the justification for read-across.

 

Read-across data:

Built on the hypothesis that Guanidine Thiocyanate dissociates in aqueous media in the corresponding ions Guanidinium and Thiocyanate, further genetic toxicity endpoints were assessed based on read-across data from Sodium Thiocyante and Guanidine Nitrate.

A detailed justification document for the read-across is attached in the respective target records of IUCID.

 

Chromosome aberration:

Sodium Thiocyanate

The ability of Sodium Thiocyanate to induce chromosome aberrations was evaluated in a study according to OECD guideline 473 in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system in two independent experiments.

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 metabolic activation.

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. Finally, it is concluded that Sodium thiocyanate is not clastogenic in human lymphocytes.

 

Guanidine Nitrate

In a mammalian cell cytogenetics assay similar to OECD Guideline 473, 1997 cultures of a clonal sub-line of a Chinese hamster fibroblast cell line (CHL) were exposed to a large number of chemicals in a comparative test.

Treatment with Guanidine Nitrate was performed at a maximum concentration of 0.5 mg/ml (41 x 10-4 M) without metabolic activation. Microscopic analysis: In the experiment no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item were close to the range of the solvent control values (< 3 % inclusive gaps).

Experimentation in the presence of metabolic activation was not performed.

Positive controls induced the appropriate response: Ethylnitrosourea and 4-Nitroquinoline-N-Oxide were among the tested substances which resulted positive and showed distinct increase in cells with structural chromosome aberrations.

In conclusion it can be stated that under the experimental conditions reported, i.e. without metabolic activation, Guanidine Nitrate did not induce structural chromosome aberrations as determined by the chromosome aberration test in CHL cells (Chinese hamster cell line) in vitro. The test item is considered to be non-clastogenic in this chromosome aberration test without metabolic activation when tested up to cytotoxic test item concentrations. There was no evidence of chromosome aberration induced over background.

Guanidine Thiocyanate

Read-across from Sodium Thiocyante and Guanidine Nitrate to Guanidine Thiocyanate was considered to be reliable without restrictions.

From both studies there is no evidence of induction of chromosome aberrations. In conclusion Guanidine Thiocyanate is considered to be not clastogenic in mammalian cells.

 

Gene mutation in mammalian cells

Sodium Thiocyanate

Mutagenic activity of Sodium thiocyanate was evaluated in an in vitro mammalian cell gene

mutation test with L5178Y mouse lymphoma cells, according to OECD guideline 476 in the  absence and presence of S9-mix.

In the first experiment, Sodium thiocyanate was tested up to concentrations of 811 μg/ml (0.01 M) in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours.

In the second experiment, Sodium thiocyanate was again tested up to concentrations of 811 μg/ml, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed at this dose level in the absence and presence of S9-mix. This is the highest concentration recommended in the guidelines.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 18- and 10-fold for MMS in the absence of S9-mix, and by 17- and 9.2-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Sodium thiocyanate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Sodium thiocyanate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Sodium thiocyanate is not mutagenic in the mouse lymphoma L5178Y test system.

 

Guanidine Nitrate

In a mammalian gene mutation assay according to OECD Guideline 476, L5178 Y (mouse lympoma thymidine kinase locus) cells cultured in vitro were exposed to guanidine nitrate (> 99.8 %) in the presence and absence of mammalian metabolic activation at the following concentrations:

Experiment I: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with and without S9 mix

Experiment II: 75.0, 150.0, 300.0, 600.0, and 1200.0 µg/mL - with S9 mix

Experiment II: 75.0, 150.0, 300.0, 600.0, and 900.0 µg/mL - without S9 mix

 

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % of survival in both parallel cultures were observed at 600 µg/mL and above in the second experiment without metabolic activation following 24 hours of treatment. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximum concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

In conclusion it can be stated that the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, Guanidinium nitrate is considered to be non-mutagenic in this mouse lymphoma assay.

Guanidine Thiocyanate

Read-across from Sodium Thiocyanate and Guanidine Nitrate to Guanidine Thiocyanate was considered to be reliable without restrictions.

From both studies there is no evidence of mutagenicity in mammalian cells. In conclusion Guanidine Thiocyanate is considered to be not mutagenic in mammalian cells.

 

Conclusion

Based on the results of the full set of in-vitro genotoxicity tests required by REACH regulation including read-across data, there is no evidence for genotoxic properties of Guanidine Thiocyanate.

Justification for classification or non-classification

A full set of in-vitro genotoxicity tests required by REACH regulation including read-across data is available. There is no evidence for a genotoxic property from gene mutation assays in bacteria and mammalian cells, as well as chromosome aberration in mammalian cells. According to CLP, EU GHS (Regulation (EC) No 1272/2008) Guanidine Thiocyanate does not need to be classified for genetic toxicity.