Ammonium 2-mercaptopropionate

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium 2-mercaptopropionate

EC Number:

236-526-4

EC Name:

Ammonium 2-mercaptopropionate

Cas Number:

13419-67-5

Molecular formula:

C3H9NO2S

IUPAC Name:

ammonium 2-sulfanylpropanoate

Test material form:

liquid

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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 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).