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Genetic toxicity in vitro

Description of key information

The in vitro genotoxicity of 1-[(2-hydroxyethyl)thio]propan-2-ol was investigated using a combination of valid testing and non-testing methods.

The in vitro genotoxicity in bacteria of the test substance was determined during a GLP study performed according to a method similar to OECD Guideline for Testing of Chemicals 471. The Ames preincubation method was performed both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range was determined during a preliminary toxicity assay and ranged between 313 and 5000 μg/plate depending on the bacterial tester strain type. As a result of this experiment, 5000 µg/plate was selected as the highest dose to be tested during the main experiment performed according to the pre-incubation method. The number of revertants induced by the test substance in each tester strain were not more than double of the solvent control value both with and without S9 mix. The reproducibility of the negative results was confirmed in dose finding test and the main test. These results led the authors of the study to the conclusion that the test substance is not mutagenic. The test item is considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.

The in vitro cytogenicity in mammalian cells of the test substance was determined during a GLP study performed according to a method similar to OECD Guideline for Testing of Chemicals 473 using Chinese lung (CHL) cell line. Cell growth inhibition was examined using 6 dose levels (156.25, 312.5, 625, 1,250, 2,500, 5,000 μg/ml) in 24- and 48-hour treatment of direct method and in metabolic activation method. Based on the results, concentrations of 1,250, 2,500, and 5,000 μg/ml for the direct and metabolic activation methods. With regard to direct method, the frequency of structural aberrant was below 1.5% in negative control group and 2.5% in exposed groups, whereas the frequency of structural aberrant was 97.5% in the positive control groups. As for metabolic activation test, the frequency of structural aberrant cells of negative control group in the absence and in the presence of S9mix was 1.0 and 1.5% respectively, while that of dosed groups showed 1.5% (with S9mix) and 1.0% (without S9mix). In contrast, positive control group showed significantly high frequency of structural aberrant cells (69.0%), compared with positive control and dosed groups. The test item is considered to be non-mutagenic under the conditions of the test.

An assessment was performed based on QSAR predictions supported by reliable experimental data on analogous substances. It concluded that 1-[(2-hydroxyethyl)thio]propan-2-ol is not expected to induce gene mutation in mammalian cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 26 July 1994 to 07 October 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Longer exposure than the one indicated in the current version of the OECD Guideline. 200 cells in metaphase analysed (300 recommended)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
other: Chinese lung (CHL) cell line
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: A cell line of fibroblast from Chinese hamster lung was obtained from the Division of Genetic and Mutagenesis, National Institute of Hygienic Sciences.
- Suitability of cells: in accordance with OECD guideline 473

MEDIA USED
Type and identity of media including CO2 concentration if applicable:
10% calf serum (inactivated by heating at 56°C for 30 minutes, lot number: 48N1141, Gibco Laboratories Co. Inc.) medium was prepared routinely from minimum essential medium of Eagle (powder form, lot number: 74K4338, Gibco Laboratories Co. Inc.).

Atmospheric condition: 5% carbon dioxide and 95% air in the humid conditions at 37°C using an automatic carbon dioxide gas cell incubator (Napco Model 6200).
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Top dose examined in the study was 5000 μg/ml. Cell growth inhibition test was performed as preliminary study (dose-finding test) using 6 dose levels (156.25, 312.5, 625, 1250, 2500, 5000 μg/ml). The cell growth rate of each exposed group was compared with that of the control group which was regarded as 100%. Because decrease of cell growth rate from 156.25 to 5000 μg/ml was not significant, 5000 μg/ml was selected as the top dose of chromosomal aberration test.
Vehicle / solvent:
Distilled water was used as vehicle for test substance.
Dimethylsulfoxide was used as vehicle for positive control.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Culture medium:
10% calf serum (inactivated by heating at 56°C for 30 minutes, lot number: 48N1141, Gibco Laboratories Co. Inc.) medium was prepared routinely from minimum essential medium of Eagle (powder form, lot number: 74K4338, Gibco Laboratories Co. Inc.).

DURATION
- Preincubation period: 5ml of medium containing 4x10^3 cells/ml was added to petri dish for 3-days incubation.
- Exposure duration: 6 hours (metabolic activation method), 24 or 48 hours (direct method)
- Expression time (cells in growth medium): 18 hours (metabolic activation method)
- Fixation time (start of exposure up to fixation or harvest of cells): not provided

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Two hours before preparing the specimens, colcemid was added to all dishes to reach a final concentration of 0.2 μg/ml. After the incubation, 2 ml 0.2% trypsin was added to dissociate the layered cells. Cells were transferred into a centrifuge tube containing 5 ml of fresh medium followed by 5-minutes centrifugation at 1000 rpm. After removal of supernatant, cells were treated with 4 ml of 75 mM potassium chloride at 37°C for 15 minutes. Furthermore, they were fixed with 1 ml cold fixative, which consisted of methanol and acetic acid in 3:1 (v/v) ratio prepared immediately before use. After the repeating of the orientation i.e., centrifugation, supernatant discard and the fixation with 4 ml of cold fixative, the cool suspension was adjusted by the fixative properly, and was dripped on two fixed spots on each slide. Slide was stained for 15 minutes with 1.4% Giemsa, which was diluted with sorensen buffer solution (pH 6.8).

NUMBER OF CELLS EVALUATED: 200 cells per group

OTHER EXAMINATIONS:
- Determination of polyploidy: The aberration of chromosome was divided into morphological and numerical. Structural chromosomal aberrations were “gaps” (of the chromatid and chromosomal types), “breaks or exchanges of the chromatid type” (of dicentric and ring chromosome, etc.) and others (fragmentation etc.). As for numerical aberration, only polyploid cells were recorded. Gap was subjected to an unstained site of chromatid where was wider than the width of chromatid and aligned on its longitudinal axis. Break would be justified when such unstained site was extremely large through it aligned on its longitudinal axis. Each aberrant category was scored, considering cells with one of these aberrations as aberrant cells.
Evaluation criteria:
In the case where statistical analysis revealed significant increase in frequencies, dose-dependence and/or the reproducibility of the test, the test substance was considered to induce chromosomal aberrations.
Statistics:
An x-square test was used to analyse frequencies of the structural aberrations including gap and the polyploid cells. When the consequence was significant, an x-square test and Fisher’s direct probability method (2x2) were applied to comparison of negative control group with each exposed group.
Key result
Species / strain:
other: Chinese lung (CHL) cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Test substance was considered to have a negative effect on chromosomal aberration of CHL cells under the provided conditions.
Executive summary:

The in vitro cytogenicity in mammalian cells of the test substance was determined during a GLP study performed according to a method similar to OECD Guideline for Testing of Chemicals 473 using Chinese lung (CHL) cell line.

Cell growth inhibition was examined using 6 dose levels (156.25, 312.5, 625, 1,250, 2,500, 5,000 μg/ml) in 24- and 48-hour treatment of direct method and in metabolic activation method. Based on the results, concentrations of 1,250, 2,500, and 5,000 μg/ml for the direct and metabolic activation methods.

With regard to direct method, the frequency of structural aberrant was below 1.5% in negative control group and 2.5% in exposed groups, whereas the frequency of structural aberrant was 97.5% in the positive control groups. As for metabolic activation test, the frequency of structural aberrant cells of negative control group in the absence and in the presence of S9mix was 1.0 and 1.5% respectively, while that of dosed groups showed 1.5% (with S9mix) and 1.0% (without S9mix). In contrast, positive control group showed significantly high frequency of structural aberrant cells (69.0%), compared with positive control and dosed groups.  

The test item is considered to be non-mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine (S. typhimurium). Tryptophan (E. coli).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
313, 625, 1250, 2500, and 5000 µg/plate (based on the result of a dose-finding test).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48h

SELECTION AGENT (mutation assays): Histidine (S. typhimurium).Tryptophan (E. coli).
Evaluation criteria:
The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The number of revertants induced by the test substance in each tester strain were not more than double of the solvent control value both with and without S9 mix. The reproducibility of the negative results was confirmed in dose finding test and the main test. These results led to the conclusion that the test substance is not mutagenic.

See attached background material.

Conclusions:
The test item is considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.
Executive summary:

The in vitro genotoxicity in bacteria of the test substance was determined during a GLP study performed according to a method similar to OECD Guideline for Testing of Chemicals 471. The Ames preincubation method was performed both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

The dose range was determined during a preliminary toxicity assay and ranged between 313 and 5000 μg/plate depending on the bacterial tester strain type. As a result of this experiment, 5000 µg/plate was selected as the highest dose to be tested during the main experiment performed according to the pre-incubation method.

The number of revertants induced by the test substance in each tester strain were not more than double of the solvent control value both with and without S9 mix. The reproducibility of the negative results was confirmed in dose finding test and the main test. These results led the authors of the study to the conclusion that the test substance is not mutagenic.

The test item is considered to be non-mutagenic under the conditions of the test, for both methods, with and without metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
other: Expert assessment
Adequacy of study:
weight of evidence
Study period:
October 05, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Assessment based on QSAR predictions supported by reliable experimental data on analogous substances
Qualifier:
no guideline required
Principles of method if other than guideline:
Assessment based on QSAR predictions supported by reliable experimental data on analogous substances
Type of assay:
other: Mouse lymphoma mammalian mutagenesis assay (in vitro)
Key result
Species / strain:
other: not applicable
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
other: not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: Assessment based on QSAR predictions supported by reliable experimental data on analogous substances

see attached document

Conclusions:
It concluded that 1-[(2-hydroxyethyl)thio]propan-2-ol is not expected to induce gene mutation in mammalian cells.
Executive summary:

An assessment was performed based on QSAR predictions supported by reliable experimental data on analogous substances. It concluded that 1-[(2-hydroxyethyl)thio]propan-2-ol is not expected to induce gene mutation 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

Justification for classification or non-classification

The capacity of 1-[(2-hydroxyethyl)thio]propan-2-ol to induce gene mutation in bacteria and mammalian cells and structural chromosomal aberration in mammalian cells was investigated by performing in vitro studies according to the relevant OECD Testing Guidelines and an expert assessment based on valid QSAR predictions supported by reliable data from an analogue substance. Results from the studies and expert assessment were negative.

In accordance with Annexes VII and VIII of REACH it was not required to consider in vivo genotoxicity testing on 1-[(2-hydroxyethyl)thio]propan-2-ol as no positive result was obtained.

As a conclusion 1-[(2-hydroxyethyl)thio]propan-2-ol does not meet the criteria for classification as mutagenic in accordance with Regulation (EC) N° 1272/2008.