[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity:

In vitro: Gene mutation test in bacteria (Ames Test): S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, up to 5000 µg/plate, plate incorporation,±S9: negative±S9 (OECD 471, GLP)

In vitro: Chromosome aberration test in mammalian cells (micronucleus test): human lymphocytes, up to 120 µg/ml,±S9: negative±S9 (OECD 487, GLP)

In vitro: Gene mutation test in mammalian cells (TK assay): mouse lymphoma cells, up to 40 µg/mL,±S9: negative±S9 (OECD 490, GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995-01-13 - 1995-02-24 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 (previous version) with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

by Umweltministerium Baden-Württemberg

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: NCTC National Collection of Type Cultures, Central Public Health Laboratory in London

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Wistar rat S9

Test concentrations with justification for top dose:

Maximum concentration: 5000 µg/plate, as stipulated by the guideline
at least 5 analyzable concentration, covering at least 2 logarithmic decades

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: good solubilizing properties

Untreated negative controls:

yes

Remarks:

aqua dest.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: min. 48 h

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: 3 plates per concentration, two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

As set out in OECD TG 471, and as stipulated under REACH

Evaluation criteria:

A test item is considered positive if either a significant dose-dependent increase in the number of revertants or a significant and reproducible increase for at least one concentration of the test item is observed.
A significant effect is described as follows:
A test item is considered mutagenic if the number of revertants is increased twice (TA 100) or trice (TA98, TA1535, TA1537) over background.
A dose-dependent increase of the number of revertant is considered also as an indication of a mutagenic potential, even if the above-mentioned increase was not reached.
Commonly valid conditions for the assessment of the results are:
- normal background growth in all agar plates
- normal ranges of spontaneous revertant rates compared with negative control groups without metabolic activation

Ranges of spontaneous revertant rates:
TA98: 15 - 60
TA100: 75 - 200
TA1535: 3 - 37
TA1537: 4 - 31

Statistics:

Of each three plates per concentration mean and standard deviation was calculated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

not valid

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

precipitation occurred at doses >= 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none stated
- Effects of osmolality: none stated
- Evaporation from medium: unknown
- Precipitation: at doses >= 500 mg/plate

RANGE-FINDING/SCREENING STUDIES:
In all plates treated with the test item a normal backgroung growth was observed. Due to precipitation at doses >= 500 mg/plate, in the main experiment the following concentrations were tested: 500, 250, 50, 25, and 5 µg/plate

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: partially exceeded, but without relevance for validity of the experiment

Conclusions:

The study was conducted under GLP according to the previous version of OECD TG 471 and is sufficiently documented, validity criteria were met. Only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible. In consequence, the results of this well-conducted study can be considered to be sufficiently reliable to assess the mutagenic potential of the test item. At none of the tested concentrations neither a significant and reproducible increase in revertants nor a dose-dependent increase of revertants at at least one concentration of the test item was observed. Hence, the test item does not need to be considered as mutagenic in bacteria.

Executive summary:

The study was conducted under GLP according to the previous version of OECD TG 471. S. typhimurium strains TA98, TA100, TA1535, TA1537 were exposed to various concentrations of the test item ranging from 2.5 - 5000 µg/plate in the plate incorporation method ±S9. Two independent experiments were performed.

No cytotoxicity was observed up to the limit dose, precipitation occurred >= 500 µg/plate. Positive and negative control gave the appropriate results. At none of the tested concentrations neither a significant and reproducible increase in revertants nor a dose-dependent increase of revertants at at least one concentration of the test item was observed. Hence, the test item does not need to be considered as mutagenic in bacteria.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-15 - 2017-09-15 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP

Qualifier:

according to guideline

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. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

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: Room temperature in the dark

OTHER SPECIFICS: Formulated concentrations were adjusted to allow for the stated water/impurity content of the test item.

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: Preliminary Toxicity Test: male, aged 29 years, Main Experiment: male, aged 28 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 rat (sprague-Dawley) liver S9

Test concentrations with justification for top dose:

The test item was considered to be a mixture and therefore the maximum recommended dose was 5000 μg/mL. The purity of the test item was accounted for in the test item formulations. Concentrations were:
0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/mL in the preliminary toxicity test
0, 10, 20, 40, 50, 60, 80 and 100 μg/mL (4h exposure, -S9)
0, 10, 20, 40, 50, 80, 100 and 120 μg/mL (4h exposure, +S9)
0, 5, 10, 20, 40, 50, 60 and 80 μg/mL (24h exposure, -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in Minimal Essential Medium at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 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 (DC)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

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

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures (A and B) were established for each dose level

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 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. 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: 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 micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration).

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: cytokinesis block proliferation index (CBPI)

Evaluation criteria:

Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9 mix
• Cell proliferation criteria in the solvent control were considered to be acceptable
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

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.

Statistics:

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: none
- Effects of osmolality: none
- Precipitation: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 312.5 μg/mL, in all three exposure groups.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 0, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL. The maximum dose was the maximum recommended dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 312.5 µg/mL, in all three exposure groups.
Hemolysis was observed following exposure to the test item at and above 156.25 µg/mL in the absence of S9 and at and above 39.06 µg/mL in the presence of S9. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. A reduced cell pellet was also noted at the washing stage at the end of the exposure period at and above 156.25 µg/mL in the absence of S9 and at and above 78.13 µg/mL in the presence of S9, indicating overall toxicity to the cell population.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 78.13 µg/mL in the 4-hour exposure, both in the presence and absence of metabolic activation (S9) and in the 24-hour exposure group. Dose levels at and above 156.25 µg/mL were completely toxic with no surviving cells on the slides. The test item induced evidence of toxicity in all of the three exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 100 µg/mL and 120µg/mL for the 4-hour exposure groups in the absence and presence of S9, respectively, and was 80 µg/mL for the 24-hour exposure group.

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 the test item to induce micronuclei in human lymphocytes The test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

Introduction: This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes in a OECD 487 study under GLP.

 

Methods: Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four 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 by toxicity. The dose levels selected for the Main Test were as follows:

 

Exposure Group	Final concentration of test item (µg/mL)
4-hour without S9	10, 20, 40, 50, 60, 80, 100
4-hour with S9 (2%)	10, 20, 40, 50, 80, 100, 120
24-hour without S9	5, 10, 20, 40, 50, 60, 80

 

Results: All vehicle (dimethyl sulphoxide) 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 did not induce any statistically significant increases in the frequency of cells with micronuclei. The test item demonstrated a very steep toxicity curve and approximately 50% cytostasis was achieved in the presence of S9 and in the 24-hour exposure in the absence of S9. Although the 4-hour exposure in the absence of S9 achieved less than optimum toxicity at the maximum dose level scored it was considered that the lack of a response in the 24-hour exposure group confirmed the absence of any response in this exposure group.

 

Conclusion: The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-16 - 2018-03-05 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, adopted 28 July 2015, Guideline No. 490 "In Vitro Mammalian Cell Gene Mutation Tests using the Thymidine Kinase Gene“.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity – In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Environmental Protection Agency, Health Effects Test Guidelines OPPTS 870.5300 “In vitro mammalian cell gene mutation test“, EPA 712-C-98-221, August 1998.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Storage Conditions: At room temperature

Target gene:

tk+/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

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 in 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.
The L5178Y cell line has been used successfully in in vitro experiments for many years. L5178Y cells are characterized by a high proliferation rate (doubling time 10 - 12 h in stock cultures) and cloning efficiency of untreated cells of usually more than 50 % both necessary for the appropriate performance of the study. The cells have a stable karyotype with a near diploid (40 ± 2) chromosome number
A low metabolic activity of cells under in vitro conditions is a disadvantage of assays with cell cultures as many chemicals develop a mutagenic potential after metabolization by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Large stocks of the cleansed L5178Y cell line are stored in liquid nitrogen in the cell bank of the laboratory allowing the repeated use of the same cell culture batch in experiments. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured two times prior to treatment. The cell cultures were incubated at 37 ± 1.5°C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % ambient air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 * 10-3 M
Aminopterin 2.0 * 10-5 M
Thymidine 1.6 * 10-3 M
Glycin 5.0 * 10-3 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0 * 10-4 M
Thymidine 1.6 * 10-3 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

0, 1.3, 2.5, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, 100.0 µg/ml (Experiment I, 4h, ±S9)
0, 0.6, 1.3, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0 µg/ml (Experiment II, 24h, -S9)
0, 1.3, 2.5, 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0 µg/ml (Experiment II, 4h, +S9)
Relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed at 39.9 µg/mL and above in the presence and absence of metabolic activation after 4 and 24 hours treatment. At 318.9 µg/mL and above the cell growth was completely inhibited in the presence and absence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Remarks:

solvent controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, purity 99.99%, the final concentration of DMSO in the culture medium was 1.0 % (v/v).

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

The solutions were prepared on the day of experiment.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1*10E7 (3*10E6 during 24 h exposure) cells/flask (80 cm² flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % during 24 h exposure)

DURATION
- Preincubation period: none
- Exposure duration: 4h or 24 h
- Expression time (cells in growth medium): expression and growth period of totally 48 h
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2 independent experiments, cells from each experimental group were seeded into 2 microtiter plates, two parallel cultures

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Rationale for test conditions:

As stipulated under REACH and OECD TG 490.

Evaluation criteria:

Evaluation of Results
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 10E6 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.

Statistics:

Statistical Analysis
A linear regression will be performed using a validated test script of "R", a language and environment for statistical computing and graphics (p < 0.05), to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together.
experimental group of culture I and II p-value calculated for the mean mutant frequencies
experiment I, without S9 mix 0.898
experiment II, without S9 mix 0.151
experiment II, with S9 mix 0.550

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

solvent controls

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Water solubility / Precipitation: No precipitation or phase separation occurred in the presence and absence of metabolic activation in the evaluated concentrations. Phase separation occurred at 318.9 µg/mL and above after 4 hours treatment with and without metabolic activation. Following 24 hours treatment, phase separation occurred at 637.8 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Relevant toxic effects indicated by a relative suspension growth (RSG) below 50% were observed at 39.9 µg/mL and above in the presence and absence of metabolic activation after 4 and 24 hours treatment. At 318.9 µg/mL and above the cell growth was completely inhibited in the presence and absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
See remarks

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. No substantial increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the corresponding solvent control was not exceeded. Hence, the substance does not need to be regarded as mutagenic in mammalian cells.

Executive summary:

The study was performed to investigate the potential of Amines, C12-14-tert-alkyl, reaction products with O,O-di-C1-14-alkyl hydrogen phosphorodithioate to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y, according to OECD TG 490, EU method B.17, and OPPTS 870.5300 under GLP.

The assay was performed in two independent experiments, using two parallel cultures each.

The first experiment was performed with and without metabolic activation and a treatment period of 4 hours. In the presence of metabolic activation the mean mutant frequency of the solvent control exceeded the upper limit of the acceptable range. Therefore, this experimental part was judged as invalid and repeated within the second experiment.

The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours, and in the presence of metabolic activation with a treatment period of 4 hours.

The maximum test item concentration of the pre-experiment (5102.0 µg/mL, UVCB substance) was chosen with respect to the OECD guideline regarding the purity of the test item.

No substantial and dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion: In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. Therefore, the test item is considered to be non-mutagenic in this mouse lymphoma thymidine kinase locus assay using the cell line L5178Y.

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.

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.