Cystine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Both key studies (OECD 473 and OECD 476) and the supporting study (Mutagenesis in E. coli) are negative.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Experiment I:
without and with metabolic activation: 2.5, 5.0 and 10.0 mM
Experiment II:
without metabolic activation: 1.25, 2.5 and 5.0 mM
with metabolic activation: 2.5, 5.0 and 10.0 mM

Vehicle / solvent:

Complete Culture Medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

EPA without metabolic activation, CPA with metabolic activation

Details on test system and experimental conditions:

A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix: 0.020, 0.039, 0.078, 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 mM.
The cultures were treated at each concentration. The selection of the concentrations used in experiments I and II was based on data from the pre-experiment. The following concentrations were used in the main experiments:
Experiment I and II:
without and with metabolic activation: 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 mM
In experiment I the cells were treated with the test item for 4 h without and with metabolic activation. The metaphases were prepared 20 h after the treatment. In experiment II, with metabolic activation, the cells were treated for 4 h and the metaphases were prepared 20 h after the treatment. In experiment II, without metabolic activation, the cells were treated for 20 h and the metaphases were prepared at the end of the treatment. The dose group selection for microscopic analyses of chromosomal aberrations was based on the mitotic index in accordance with the guidelines.
The following concentrations were selected in the main experiments for the microscopic analyses:
Experiment I: without and with metabolic activation: 2.5, 5.0 and 10.0 mM
Experiment II: without metabolic activation: 1.25, 2.5 and 5.0 mM; with metabolic activation: 2.5, 5.0 and 10.0 mM

Exposure time 4 hours (without and with S9 mix; first experiment with S9 mix;
second experiment):
Two days after seeding of the cells, the culture medium was replaced with serumfree medium containing the test item and S9 mix (with metabolic activation). Additional negative and positive controls were performed without and with exogenic metabolic activation.
4 h after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
Exposure time 20 hours (without S9 mix; second experiment):
Two days after seeding of the cells the culture medium is replaced with complete medium containing the test item. This medium is not changed until preparation of the cells. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.
All cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0% CO2 (95.0% air).

Preparation of the Cultures
Colcemid® was added to the cultures (0.2 μg/mL culture medium) 17.5 hours after the start of each treatment (4 h and 20 h treatment). 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4% KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid (v/v). All the steps were carried out on precision hot plates at 37 °C. After fixation step the slides were air dried and stained with Giemsa.

Evaluation criteria:

The chromosomal aberration assayis considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.0% (without and with metabolic activation),
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item L-Cysteine is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of L-Cysteine (99.6% pure) to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay according to OECD Guideline 473 was carried out.

The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 hwithoutandwithmetabolic activation in experiment I. In experiment II, the treatment interval was 20 hwithoutand 4 hwithmetabolic activation. Parallel cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:withoutandwithmetabolic activation: 2.5, 5.0 and 10.0 mM

Experiment II:withoutmetabolic activation: 1.25, 2.5 and 5.0 mM;withmetabolic activation: 2.5, 5.0 and 10.0 mM

No precipitation of the test item was notedwithoutandwithmetabolic activation in all dose groups evaluated in experiment I and II. No cytotoxic effects of the test item were notedwithoutandwithmetabolic activation in all concentrations evaluated in experiment I and II. In experiment Iwithoutandwithmetabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control.

In experiment IIwithoutmetabolic activation a slight increase of aberrant cells (4.5%) was noted compared to the historical control data (0.0% - 4.0%). However, as there was no concentration relationship observed and only one of the four evaluated slides displayed an increase (2%, 1%, 3% and 12% aberrant cells observed) this effect was considered as not biologically relevant.

In experiment IIwithmetabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. In the experiments I and IIwithoutandwithmetabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls.

In the experiments I and IIwithoutandwithmetabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls.

EMS (400 and 900μg/mL) and CPA (0.83μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects. In the first experimentwithoutmetabolic activation the positive control EMS (900μg/mL) displayed a percentage of 5% aberrant cells.

However, on one of the two slides additional five aberrations were seen in metaphases with less than 21 chromosomes. This would lead to an aberration rate of 7.5%.

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item LCysteine did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item L-Cysteine is considered to be non-clastogenic in this chromosome aberration test.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Key studies:

To investigate the potential of L-Cysteine (99.6% pure) to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay according to OECD Guideline 473 was carried out. The metaphases were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 20 h without and 4 h with metabolic activation. Parallel cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations: Experiment I: without and with metabolic activation: 2.5, 5.0 and 10.0 mM. Experiment II: without metabolic activation: 1.25, 2.5 and 5.0 mM; with metabolic activation: 2.5, 5.0 and 10.0 mM. No precipitation of the test item was noted without and withmetabolic activation in all dose groups evaluated in experiment I and II. No cytotoxic effects of the test item were noted without and with metabolic activation in all concentrations evaluated in experiment I and II. In experiment I without and with metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control. In experiment II without metabolic activation a slight increase of aberrant cells (4.5%) was noted compared to the historical control data (0.0% - 4.0%). However, as there was no concentration relationship observed and only one of the four evaluated slides displayed an increase (2%, 1%, 3% and 12% aberrant cells observed) this effect was considered as not biologically relevant. In experiment II with metabolic activation no biologically relevant increase of the aberration rates was noted after treatment with the test item. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls. In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the negative controls. EMS (400 and 900μg/mL) and CPA (0.83μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects. In the first experiment without metabolic activation the positive control EMS (900μg/mL) displayed a percentage of 5% aberrant cells. However, on one of the two slides additional five aberrations were seen in metaphases with less than 21 chromosomes. This would lead to an aberration rate of 7.5%. In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item LCysteine did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item L-Cysteine is considered to be non-clastogenic in this chromosome aberration test.

The test item L-Cysteine was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster according to OECD Guideline 476. The selection of the concentrations was based on data from the pre-experiments. In experiment I and II 10 mM (with and without metabolic activation) was selected as the highest concentrations. Experiment I with and without metabolic activation and experiment II with metabolic activation were performed as a 4 h short-term exposure assay. Experiment II without metabolic activation was performed as 20 h long time exposure assay. The test item was investigated at the following concentrations: Experiment I without and withoutmetabolic activation: 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 mM. Experiment II withoutmetabolic activation: 0.010, 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0 and 10 mM and with metabolic activation: 0.07, 0.1, 0.4, 0.7, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 mM. No precipitation of the test item was noted in the experiments. A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II without metabolic activation. No biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment I and II with metabolic activation. In experiment I with outmetabolic activation the relative growth was 36.8% for the highest concentration (10 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 10 mM with a relative growth of 92.8%. In experiment II without metabolic activation the relative growth was 16.6% for the highest concentration (10 mM) evaluated. The highest concentration evaluated with metabolic activation was 10 mM with a relative growth of 137.8%. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item L-Cysteine is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Additionally literature is available for Bacterial Reverse Mutation tests performed with the read-across substance L-Cysteine and N-Acetyl-Cysteine. Testing results are negative for N-Acetyl-Cysteine and ambigious for most of the L-Cysteine tests. The reasons for ambigious results have been extensively studied by H. Glatt und F. Oesch and it could be explained why only several strains (TA 100 and also TA 92) are especially sensitive to sulfur-containing compounds. These effects produced with sensitive strains in in-vitro systems are not considered relevant for classification and the overall key result of Bacterial Reverse Mutation tests is asumed to be negative.

Justification for selection of genetic toxicity endpoint
Guideline study (OECD 473) under GLP conditions. There is also another Klimisch 1 study available (HPRT test, OECD 476) which could equally
be used as endpoint. No adverse effect was observed in either study.

Justification for classification or non-classification

L-Cystine does not need to be classified. The read-across is justified due to the rapid conversion of L-Cysteine to L-Cystine and due to the fact that in many proteins two molecules of L-Cysteine form a disulfide bridge resulting in L-Cysteine. The mechanisms are described in detail in other sections of this CSR.