Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro: Gene mutation (Bacterial reverse mutation, Ames test): S. typhimurium TA 98, TA 100 and E. coli WP2 uvr A pKM 101: negative with and without metabolic activation

Genetic toxicity in vitro: Gene mutation (mouse lymphoma assay), OECD 490, GLP: mouse lymphoma L5178Y cells: negative with and without metabolic activation

Genetic toxicity in vitro: Chromosome aberration (Micronucleus test), OECD 487, GLP: human lymphocytes: negative with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Protocol of the National Toxicology Program (NTP), performed on strains TA100, TA98, eColi pKM101
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
his-
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Remarks:
cited as eColi pKM101
Metabolic activation:
with and without
Metabolic activation system:
induced male Sprague Dawley rat liver S9 (RLI)
Test concentrations with justification for top dose:
0, 25, 50, 100, 200, 250, 400, 500, 750, 1000, 2000, 3500, 5000, or 7500 µg/plate (TA100, TA98)
0, 25, 50, 100, 150, 200, 250, 400, 500, 750, or 1000 µg/plate (eColi pKM101)
Concentrations chosen based on cytotoxicity.
A minimum of five dose levels covering a range of at least three logs should be selected for the definitive test. Two or three plates should be used for each dose level and for the controls. For toxic chemicals, only the highest dose used should exhibit toxicity. For non-toxic chemicals, a high dose of 5000 or 10,000 mg/plate is acceptable.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-Nitro-o-phenylenediamine (2.5 µg/plate) -S9 / 2-Aminoanthracene (1-5 µg/plate) +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days

SELECTION AGENT (mutation assays): his minimal agar

- OTHER: Method as cited in
K. Mortelmans, E. Zeiger / Mutation Research 455 (2000) 29–60
https://ntp.niehs.nih.gov/testing/types/genetic/index.html

Multiple sets of cultures are prepared using a range of doses and different amounts of liver enzymes and bacteria strains. Each culture is prepared in a test tube containing a suspension of one bacterial strain and either an S9 mix or a plain buffer. Then the test substance is added. Control cultures are created with the same ingredients, but without the test substance. A positive control group is created using a known mutagen. Once prepared, each culture is incubated for 20 minutes at 37º C.
After the first incubation period, agar is mixed into the cultures, and each tube is poured onto the surface of a Petri dish prepared with the standard medium. The plates are then incubated, usually for two days.
Rationale for test conditions:
as cited in K. Mortelmans, E. Zeiger / Mutation Research 455 (2000) 29–60
Evaluation criteria:
The test is only valid if the number of positive control colonies is greater than for the control cultures. Once this has been confirmed, the test cultures are compared to the control. If the substance is mutagenic, it will have caused greater bacteria growth.
• A positive response is a reproducible, dose-related increase in any set of test cultures. There is no minimum percentage of increase to define a result as positive.
• An equivocal response is any increase that is not reproducible, not dose-related, or not statistically significant.
• A negative response occurs when none of the cultures tested shows more growth than the control.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The study was performed equivalent to OECD TG 471 with minor deficiencies in documentation and performance.
Testing was performed on bacterial strains S. typhimurium TA100, TA98, and E. coli WP2 uvr A pKM 101. Those strains detect the following mutations:
TA100: transitions and transversions, some frameshifts (base substitution at G:C basepair)
TA98: Frameshifts at (or near) GCGCGCGC (DeltaGpC frameshift)
E. coli WP2 uvr A pKM: has an AT base pair at the primary reversion site and may detect certain oxidising mutagens, cross-linking agents and hydrazines, which may not be detected by the four S. typhimurium strain in OECD TG 471 of 1981.
According to the recent OECD TG 471, the following 5 strains should be used: S. typhimurium TA1535, and S. typhimurium TA1537 or TA97 or TA97a, and S. typhimurium TA98, and S. typhimurium TA100, and E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. The lacking two strains detect the following mutations:
TA1535: Transitions and transversions (base substitution at G:C basepair)
TA1537 or TA97: Frameshifts at (or near) GGGGG (TA1537) or GGGGGG (TA97)
As obvious, the mutations detected by the lacking TA1535 strain are similar to the ones detected by TA100, which in addition detects some frameshift, so TA100 may serve as a surrogate for TA1535. Further, due to its ability to detect at least some frameshifts at the G:C basepair, the obtained negative result may give some indication that testing on TA1537 or TA97 on G-rich sites would also reveal a negative result, too. Overall, the used three strains are well-selected to cover the broadest possible spectrum of mutations with the least efforts, so the deviations from the recent OECD 471 can be considered as minimal and give a first indication on the mutagenic potential of 2,5-Dimercapto-1,3,4-thiadiazole, which will be further evaluated via a higher tier mammalian cell mutation test (OECD 490).
Positive and negative controls gave the appropriate results. Hence, the results can be considered to be sufficiently reliable to assess the potential of 2,5-Dimercapto-1,3,4-thiadiazole to induce gene mutations in bacteria. In all tested strains, 2,5-Dimercapto-1,3,4-thiadiazole did consistently not induce gene mutations, the outcome of the test can be considered negative.
This outcome is further supported by the results of the available OECD 490 study: Therein, the test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, limited by both toxicity and precipitation, in any of the three exposure groups. So there is still consistently shown that the test item is incapable to induce gene mutation, both in bacteria and mammalian cells, so the lack of the bacterial strains TA1535 and TA1537 or TA97 does clearly not underestimate the potential hazard of 2,5-Dimercapto-1,3,4-thiadiazole, and the present Ames test is sufficient to cover the endpoint gene mutation in bacteria, no further tests need to be conducted.
Executive summary:

In a reverse gene mutation assay in bacteria (similar to OECD TG 471), strains S. typhimurium TA100, TA98, and E. coli WP2 uvr A pKM 101 were exposed to 2,5-Dimercapto-1,3,4-thiadiazole in DMSO at concentrations of max. 7500 µg/plate in the presence and absence of mammalian metabolic activation (induced rat liver S9) via the pre-incubation method.

2,5-Dimercapto-1,3,4-thiadiazole was tested up to cytotoxic concentrations. The positive and negative controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-02-28 - 2017-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals (2014) No. 487 "In Vitro Mammalian Cell Micronucleus Test", adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C in the dark
Target gene:
n/a
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- Cell cycle length, doubling time or proliferation index: Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
- Sex, age and number of blood donors if applicable:
The details of the donors used are:
Preliminary Toxicity Test: male, aged 33 years
Main Experiment: male, aged 30 years
- Whether whole blood or separated lymphocytes were used if applicable: whole blood cultures

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
PB/βNF induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
The molecular weight of the test item was given as 150.24568, therefore, the maximum recommended dose level was 1500 μg/mL. The purity of the test item was 97.34% and was accounted for in the test item formulations.
The test item was insoluble in aqueous media at 15 mg/mL but was soluble in DMSO at 150 mg/mL in solubility checks performed in-house.
Pre-Test: The dose range of test item used was 0, 11.72, 23.44, 46.88, 93.75, 187.5, 375, 750, 1125, 1500 μg/mL.
Main test: The dose range of test item used for all three exposure groups was 0, 12, 24, 48, 96, 192 and 384 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for the 4-hour exposure groups and both precipitate and toxicity for the 24-hour exposure group.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 15 mg/mL but was soluble in DMSO at 150 mg/mL in solubility checks performed in-house.
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (DEME-C)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: approx. 48 h
- Exposure duration: 4 or 24 h
- Expression time (cells in growth medium): 24 h with Cytochalasin B
- Fixation time (start of exposure up to fixation or harvest of cells): 28 or 48 h

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: duplicate cultures, three exposure groups in the main experiment (4h, ±S9; 24, -S9)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cell Harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of Microscope Slides: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining: When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
Cytokinesis Block Proliferation Index (CBPI): A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

DETERMINATION OF CYTOTOXICITY
- Method: other: Incidence of Hemolysis
Rationale for test conditions:
as stipulated by the guideline
Evaluation criteria:
Data Evaluation
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.
Statistics:
Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation: A precipitate of test item was noted at 384 μg/mL in all three exposure groups of the main test.
- Definition of acceptable cells for analysis:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 11.72 to 1500 μg/mL. The maximum dose was the maximum recommended dose level.
A precipitate of the test item was observed in the blood cultures at the end of the exposure at and above 187.5 μg/mL in all three exposure groups. Additionally, an increased pellet was observed at and above 187.5 μg/mL in the 24-hour exposure group only due to the amount of precipitate in the blood pellet.
Hemolysis was observed following exposure to the test item at and above 187.5 μg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 750 μg/mL in the presence of S9. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any cytotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 750 μg/mL in all three exposure groups. The test item induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for the 4-hour exposure groups and both precipitate and toxicity for the 24-hour exposure group.
The dose levels of the controls and the test item are given in the table below:
Group Final concentration of test item (µg/mL)
4-hour without S9 0*, 12, 24, 48, 96*, 192*, 384*, MMC 0.2*
4-hour with S9 (2%) 0*, 12, 24, 48, 96*, 192*, 384*, CP 5*
24-hour without S9 0*, 12, 24, 48*, 96*, 192*, 384, DC 0.075*
* = Dose levels selected for analysis of micronucleus frequency in binucleate cells
MMC = Mitomycin C
CP = Cyclophosphamide
DC = Demecolcine

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
- Indication whether binucleate or mononucleate where appropriate: The qualitative assessment of the slides determined that the precipitate and toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item (384 µg/mL) in all three exposure groups.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:The positive control items induced statistically significant increases in the frequency of cells with micronuclei.
- Negative (solvent/vehicle) historical control data: The vehicle control cultures had frequencies of cells with micronuclei within the expected range.
The response observed in the 4-hour exposure group was reduced but still statistically significant despite the response being at the upper limit of the in-house historical controls. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: hemolysis and a reduced pellet were also observed at 384 µg/ml in the 24-hour exposure group only indicating that maximum exposure to the test item had occurred.
In the 4-hour exposure group in the absence of S9, 42% cytostasis was observed at 384 μg/mL. This is below the minimum range for toxicity as stated in the OECD 487 guideline is 55±5%. Therefore, the presence of precipitate was used to select the maximum dose level for binucleate analysis which was 384 μg/mL.
In the 4-hour exposure group in the presence of S9, 34% cytostasis was achieved at 384 μg/mL. Again, optimum toxicity (55±5%) was not achieved so the presence of precipitate was used to select the maximum dose level selected for binucleate analysis (384 μg/mL).
In the 24-hour exposure group, 44%, 50% and 73% cytostasis was achieved at 96, 192 and 384 μg/mL, respectively. Therefore, 192 μg/mL was selected as the maximum dose level for binucleate analysis because this met the optimum toxicity as specified by the test guideline. The dose level 384 μg/mL was considered to be too toxic as the cytostasis value was in excess of the optimum range of toxicity.

For raw data, see attachment.

Conclusions:
The study was conducted under GLP according to OECD guideline 487 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of 2,5-Dimercapto-1,3,4-thiadiazole to induce micronuclei in mammalian cells. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 2,5-Dimercapto-1,3,4-thiadiazole is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Executive summary:

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of 2,5-Dimercapto-1,3,4-thiadiazole on the nuclei of normal human lymphocytes according to OECD TG 487 under GLP. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on primarily precipitate but the potential toxicity of the test item at precipitating dose levels was also considered. The dose levels selected for the Main Test were as follows:

Group

Final concentration of test item 2,5-Dimercapto-1,3,4-thiadiazole

4-hour without S9

0, 12, 24, 48, 96, 192, 384 µg/ml

4-hour with S9 (2%)

24-hour without S9

Results

All vehicle (dimethyl sulphoxide (DMSO)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was moderately toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI or was the lowest precipitating dose level.

Conclusion

The test item 2,5-Dimercapto-1,3,4-thiadiazole was considered to be non-clastogenic and non‑aneugenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-10-23 - 2017-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4°C, in the dark
Target gene:
tk +/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Remarks:
L5178Y TK+/- 3.7.2c
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: 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, Brighton, 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 cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 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 R10 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), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Properly maintained: Yes. The stocks of cells are stored in liquid nitrogen at approximately -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 R10 media) at 37°C with 5% CO2 in air.
- Periodically checked for Mycoplasma contamination: Master stocks of cells were tested and found to be free of mycoplasma.
- Periodically 'cleansed' against high spontaneous background: Yes. 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 R10 medium.
Metabolic activation:
with and without
Metabolic activation system:
PB/βNF induced male Sprague-Dawley rat liver S9
Test concentrations with justification for top dose:
The molecular weight of the test item was 150.24568 therefore, the maximum proposed dose level was 1500 µg/mL, the 10mM limit dose level. The purity of the test item was accounted for in the test item formulations.
Top dose was limited by toxicity and/or precipitation.
4h, -S9: 0, 12.5, 25, 50, 100, 125, 150, 175, 200 µg/ml
4h, +S9: 0, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50 µg/ml
24h, -S9: 0, 3.13, 6.25, 12.5, 25, 37.5, 50, 75, 100 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Following solubility checks performed in-house for the Human Lymphocyte in vitro Micronucleus Test performed on the same test item, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.
Untreated negative controls:
yes
Remarks:
solvent controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable):
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9.
The cells were counted and processed to give 1 x 10E6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10E6 cells/mL in 10 mL cultures were established in 25 cm² tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation

DURATION
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days.
- Selection time (if incubation with a selection agent): ten to twelve days

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate cultures (A and B), three independent conditions

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth (%RSG)
Rationale for test conditions:
as set out in the guideline
Evaluation criteria:
Due to limitations of this free-text field, see "Any other information on materials and methods"
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
top dose was also limited by precipitation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant pH shift noted in the evaluated concentration range
- Effects of osmolality: no relevant osmolality shift noted in the evaluated concentration range
- Precipitation: yes, at top dose

RANGE-FINDING/SCREENING STUDIES:
There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all of the three exposure groups when compared to the concurrent vehicle control groups. Test item precipitate was observed at and above 200 µg/mL in the 4-hour exposure group in the absence of metabolic activation, at and above 25 µg/ml in the 4-hour exposure group in the presence of metabolic activation, and at 400 µg/ml in the 24-hour exposure group in the absence of metabolic activation, at the end of the exposure periods. Therefore, the maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item induced toxicity in the 4-hour exposure group in the absence of metabolic activation, the onset of test item precipitate in the 4-hour exposure group in the presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
For details, see tables below.
Conclusions:
The study was conducted under GLP according to OECD guideline 490 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the potential of the test item to induce mutations in mammalian cells. The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction: The GLP study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

 

Methods: One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (DMSO), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

 

Mutagenicity Test

Group

Concentration of DMTD (CAS No. 1072-71-5) (µg/mL) plated for viability and mutant frequency

4-hour without S9

12.5, 25, 50, 100, 125

4-hour with S9 (2%)

1.56, 3.13, 6.25, 12.5, 25, 50

24-hour without S9

3.13, 6.25, 12.5, 25, 37.5, 50

 

Results:The maximum dose levels in the subsequent Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item‑induced toxicity in the 4-hour exposure group in the absence of metabolic activation, the onset of test item precipitate in the 4-hour exposure group in the presence of metabolic activation, and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

Conclusion: The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

As all available and required in vitro genotoxicity tests, i.e. testing for gene mutations in both bacteria and mammalian cells as well as chromosome mutations in mammalian cells, revealed negative results, no conclusion on a mode of action for genotoxic events can be drawn. No indication is given that the obtained results are not relevant for humans, as in vivo metabolism of the test item is sufficiently mimicked by addition of S9 mix, human lymphocytes were also tested and direct genotoxins act commonly species-independent.

Additional information

Justification for classification or non-classification

All available test results for gene mutation and chromosome aberrations (micronucleus test) in vitro are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified. Gene and chromosome mutations are considered initial steps in rather complex carcinogenesis. As the substance does not induce those, no need to consider the substance as carcinogen is evident.

According to Regulation 1272/2008 and amendments, regarding “3.5. Germ cell mutagenicity“, this hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class. Further, substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens. Last but not least, to arrive at a classification, test results are considered from experiments determining mutagenic and/or genotoxic effects in germ and/or somatic cells of exposed animals. Mutagenic and/or genotoxic effects determined in in vitro tests shall also be considered.

In consequence, consistent negative results in three different in vitro test systems addressing three different events related to genetic damage allow to conclude that the substance does not need to be classified as germ cell mutagen.