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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 01, 1995 to April 19, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
see 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
other: EEC Directive 92/69, L 383 A
Deviations:
yes
Remarks:
see 'Principles of method if other than guideline'
Principles of method if other than guideline:
a) The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar Hanibm (BRL, CH-4414 Fiillinsdorf; body weight approx. 220 - 320 g).
b) "Chemikaliengesetz ("Chemicals Act") of the Federal Republic of Germany, Anlage 1 ("Annex l"), dated July 25, 1994 (BGBL I S. 1703).
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Test concentrations with justification for top dose:
The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Blood Collection and Delivery:

For this study (for both experiments) blood was collected only from a single donor (male; age: 34 years) to reduce inter individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. med. V. Theodor (Odenwaldring 4, 64380 Rofldorf). The tubes were sent to CCR to initiate cell cultures within 24 hours after blood collection. Before use the blood was stored under sterile conditions at 4 °C.

Mammalian microsomal fraction S9 mix:

S9 (Preparation by CCR):

The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male rats, strain Wistar HanIbm in deviation to the protocol (BRL, CH-4414 Flillinsdorf; body weight approx. 220 -320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Karlsruhe) in olive oil 5 days previously.
After cervical dislocation, the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged twice at 9,000 g for 10 minutes (4 °C).
A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 3 ml and stored at -70 °C. Small numbers of the ampoules were kept at -20 °C for only several weeks before use. The protein content was determined using the analysis kit of Bio-Rad. Laboratories, D-80939 Munchen.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml. The protein concentration of the S9 used was 26.2 mg/ml.

S9 mix:

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.74 mg/ml in the cultures. Cofactors were added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-orthophosphate buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
Evaluation criteria:
A test substance is classified as mutagenic if it induces reproducibly either a concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test substance producing reproducibly neither a concentration­related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Without S9 mix the reduction of the mitotic· index was less pronounced (solvent control = 100 %, 1000.0 µg/ml = 74.1 %). However, preparation of higher concentrations could not be evaluated due to low mitotic index combined with poor metaphase quality.
- In the absence as well as in the presence of metabolic activation the substance did not increase the frequency of structural chromosomal aberrations as compared to the frequency of the corresponding solvent controls. Biometric evaluation of the results was done by means of the chi-square test. The aberration frequencies after treatment with the test substance proved not to be statistically significant different from the solvent control frequencies.
- EMS (440.0 µg/ml) and CPA (45.0 µg/ml) were used as positive controls and induced statistically significant increases in cells with structural chromosomal aberrations.
- The proliferation index of the lymphocytes in solvent control cultures was checked by analyzing the proportion of mitotic cells in the 1st, 2nd and 3rd metaphase (Ml, Ml+, M2 and M3) and showed that the lymphocytes divided adequately within the fixation interval.
- The test substance induced no structural chromosomal aberrations in human lymphocytes in vitro.
Conclusions:
Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test.
Executive summary:

A study was conducted to determine the genotoxic potential of the test substance (Ammonium 2-mercaptopropionate) to induce formation of micronuclei in human lymphocytes cultured in vitro, according to OECD Guideline 473 and EEC Directive 92/69, L 383 A, in compliance with GLP. The experiment was performed with a preparation time of 22 h after treatment with the test substance which was dissolved in culture medium. In each experimental group two parallel cultures were analyzed. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL. In the absence as well as in the presence of S9 mix, the test substance was tested up to cytotoxic concentrations. Appropriate reference mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosomal aberrations. Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test (Czich, 1995).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 01, 2007 to May 04, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Preliminary test: 4.81 to 1232 µg/ml,
Experiment 1: 154 to 1232 µg/ml,
Experiment 2: 77 to 1232 µg/ml in the absence of S9-mix and 154 to 1232 µg/ml in the presence of S9-mix.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Cell Line:
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture:
The stocks of cells are stored in liquid nitrogen at -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10 % donor horse serum (giving RIO media) at 37 °C with 5 % CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20 % donor horse serum (R20) and without serum (RO) are used during the course of the study.

Cell Cleansing:
The TK heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/ml), Hypoxanthine (15 µg/ml), Methotrexate (0.3 µg/ml) and Glycine (22.5 µg/ml). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to RIO medium.

Preparation of test and control substance:
The test substance was accurately weighed and diluted with R0 medium. The maximum concentration level was 1232 µg/ml, which was equivalent to 10 mM. The content of the active ingredient was 70.0 % and was accounted for in the formulations. There was no marked change in pH when the test substance was dosed into media and the osmolality did not increase by more than 50 mOsm.

Metabolic activation:
PB/βNF S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing approx. 250 g.
Rationale for test conditions:
Results from the preliminary toxicity test were used to set the test substance concentration levels for the mutagenicity experiments. Maximum concentration levels were selected using the following criteria:
i) Maximum recommended concentration level, 5000 µg/ml or 10 mM.
ii) The presence of excessive precipitate where no test substance-induced toxicity was observed.
iii) Test substance-induced toxicity, where the maximum concentration level used should produce 10 to 20 % survival (the maximum level of toxicity required).
Evaluation criteria:
For a test substance 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 substance concentration level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126-E6 will be considered positive. However, if a test substance produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a concentration-related response, then it may be considered to have no toxicological significance.
Statistics:
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:

In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was no marked reduction in the %RSG of cells treated with the test substance when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was a clear concentration-related reduction of the %RSG values of cells treated with test substance indicating optimum levels of toxicity at the 1232 µg/ml concentration level.

Experiment 1:

- There was no evidence of toxicity following exposure to the test material in the 4-hour exposure group in the presence of metabolic activation and only a slight reduction in the 4-hour exposure group in the absence of metabolic activation as indicated by the %RSG and RTG values.
- The test substance did not induce any statistically significant or concentration related (linear-trend) increases in the mutant frequency in either the absence or presence of metabolic activation.

Experiment 2:

- The test substance did not induce any statistically significant or concentration-related increases in the mutant frequency in the 4-hour exposure group in the presence of metabolic activation.
- In the 24-hour exposure group in the absence of metabolic activation, the test substance induced a small but statistically significant increase in the mutant frequency at the upper surviving concentration level. However, the increase did not exceed the GEF value and was at a concentration level approaching the toxic limit. Therefore, it was considered that the response was due to a cytotoxic rather genotoxic mechanism and was, therefore, of no toxicological significance. This interpretation is supported by the fact that at a higher concentration of the test substance after treatment for 4-hours (with or without metabolic activation) an increase in induced mutations was not observed in the absence of marked cytotoxicity.

In Experiment 1 and 2:
- Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200-E6 viable cells. Both of 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.
- Precipitate of test substance was not observed at any of the concentration levels.
Conclusions:
Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells.
Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance (Ammonium 2-mercaptopropionate) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD 476 Guideline and EU Method B.17, in compliance with GLP. Two independent experiments were performed. The exposure groups were 4-hours in the presence of metabolic activation and 24-hours in the absence of metabolic activation. The concentration range of the active ingredient was selected following the results of a preliminary toxicity test and was 154 to 1232 µg/mL for the first experiment. For the second experiment the concentration range of the active ingredient was 154 to 1232 µg/mL with activation and 77 to 1232 µg/mL without activation. The maximum concentration level used, 1232 µg/mL, was the 10 mM limit concentration. Precipitate of test substance was not observed at any of the concentration levels. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any concentration level, either with or without metabolic activation, in either the first or the second experiment at concentration levels up to and including the 10 mM limit concentration. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2007).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 22, 1996 to March 29, 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
no
Remarks:
Internal QA of the quality of the bacterial strain used for testing.
Type of assay:
bacterial forward mutation assay
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: carries the hisG gene (his G46 mutation)
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: carries the hisD gene (hisD 3052 mutation)
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: carries the hisG gene (his G46 mutation)
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: carries the hisD gene (hisD 3052 mutation)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver extract (S9 fraction)
Test concentrations with justification for top dose:
0.02, 0.05, 0.1, 0.2 and 0.5 µl thiolactic acid/plate. Toxic to slightly toxic effects observed at 0.5 µl/plate in the main experiment.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: aminoanthracene
Remarks:
Positive controls tested with S9 rat liver fraction only (main experiment)
Key result
Species / strain:
S. typhimurium TA 98
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:
valid
Key result
Species / strain:
S. typhimurium 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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
valid
Key result
Species / strain:
S. typhimurium TA 1538
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:
valid
Additional information on results:
See results tables attached under 'Attached background material'.
Conclusions:
Under the conditions of the Ames study, the test substance was not mutagenic.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 using the plate incorporation method. The tester strains were Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1538. Testing was carried out twice at a 7 day interval using 5 concentrations ( 0.02, 0.05, 0.1, 0.2 and 0.5 µl thiolactic acid/plate) and 3 runs per dose, in the presence and absence of a rat liver metabolic activation system (S9 fraction). The experiment included a negative control (no test substance) and two positive control substances (aminoanthracene for TA 98 and TA 1538) and sodium azide (for TA 100 and TA 1535)). The highest selected concentration (0.5 µl/plate) was toxic to slightly toxic to the bacteria in the main experiment. The spontaneous rate of the selected tester strains was within normal variation. The revertant rate in the presence of the test substance was within the spontaneous rate at all concentrations, with and without S9 mix. Under the conditions of the Ames study, the test substance was therefore not mutagenic (Riess, 1996).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro

Study 1:

A study was conducted to determine the mutagenic potential of the test substance according to OECD Guideline 471 using the plate incorporation method. The tester strains were Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1538. Testing was carried out twice at a 7 day interval using 5 concentrations ( 0.02, 0.05, 0.1, 0.2 and 0.5 µl thiolactic acid/plate) and 3 runs per dose, in the presence and absence of a rat liver metabolic activation system (S9 fraction). The experiment included a negative control (no test substance) and two positive control substances (aminoanthracene for TA 98 and TA 1538) and sodium azide (for TA 100 and TA 1535)). The highest selected concentration (0.5 µl/plate) was toxic to slightly toxic to the bacteria in the main experiment. The spontaneous rate of the selected tester strains was within normal variation. The revertant rate in the presence of the test substance was within the spontaneous rate at all concentrations, with and without S9 mix. Under the conditions of the Ames study, the test substance was therefore not mutagenic (Riess, 1996).

Study 2:

A study was conducted to determine the genotoxic potential of the test substance (Ammonium 2-mercaptopropionate) to induce formation of micronuclei in human lymphocytes cultured in vitro, according to OECD Guideline 473 and EEC Directive 92/69, L 383 A, in compliance with GLP. The experiment was performed with a preparation time of 22 h after treatment with the test substance which was dissolved in culture medium. In each experimental group two parallel cultures were analyzed. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated, without S9 mix (exposure period 22 h): 100.0; 300.0; 1000.0 µg/mL and with S9 mix (exposure period 4 h): 300.0; 1000.0; 3000.0 µg/mL. In the absence as well as in the presence of S9 mix, the test substance was tested up to cytotoxic concentrations. Appropriate reference mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosomal aberrations. Under the study conditions, the test substance did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-mutagenic in this chromosome aberration test (Czich, 1995).

Study 3:

A study was conducted to determine the potential mutagenicity of the test substance (Ammonium 2-mercaptopropionate) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line according to OECD 476 Guideline and EU Method B.17, in compliance with GLP. Two independent experiments were performed. The exposure groups were 4-hours in the presence of metabolic activation and 24-hours in the absence of metabolic activation. The concentration range of the active ingredient was selected following the results of a preliminary toxicity test and was 154 to 1232 µg/mL for the first experiment. For the second experiment the concentration range of the active ingredient was 154 to 1232 µg/mL with activation and 77 to 1232 µg/mL without activation. The maximum concentration level used, 1232 µg/mL, was the 10 mM limit concentration. Precipitate of test substance was not observed at any of the concentration levels. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system. The test substance did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any concentration level, either with or without metabolic activation, in either the first or the second experiment at concentration levels up to and including the 10 mM limit concentration. Under the study conditions, the test substance was considered to be non-mutagenic to L5178Y cells (Flanders, 2007).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not require classification for mutagenicity according to EU CLP (EC 1272/2008) criteria.