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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG471): not mutagenic

Chromosome aberration (OECDTG473): not clastogenic

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:
20 June 2019 - 15 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.
Test concentrations with justification for top dose:
Direct plate assay:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 0.18, 0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study:
TA1535, without S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
TA1537 and TA98, without S9-mix: 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
TA1535, TA1537 and TA98, with S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate

Pre-incubation assay:
Experiment 1
Preliminary test, without S9, TA100 and WP2uvrA: 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
Preliminary test, with S9, TA100: 0.18, 0.55, 1.7, 5.4, 17, 52, 164 and 512 μg/plate
Preliminary test, with S9, WP2uvrA: 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate
Main study:
TA1535, TA1537 and TA98, without S9-mix: 0.056, 0.18, 0.55, 1.7, 5.4 and 17 μg/plate
TA1535, TA1537 and TA98, with S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear (light yellow to colourless) solution in ethanol.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other:
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA) in DMSO, 1 μg for TA98 and TA100 (Direct plate assay), 2.5 μg for TA1535 and TA1537, 5 μg for TA100 (Pre-incubation assay), 15 μg for WP2uvrA
Remarks:
With S9
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
Not performed.
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
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle 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:
cytotoxicity
Vehicle controls validity:
valid
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the substance on the plates was not observed at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES (if applicable): Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both TA100 and WP2uvrA tester strains in the absence and presence of S9-mix. Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test item was tested up to concentrations of 164 μg/plate in the absence of S9-mix in both tester strains and up to 512 and 1600 μg/plate in tester strains TA100 and WP2uvrA, respectively, in the presence of S9-mix. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the first mutation experiment, the test item was tested up to concentrations of 164 and 512 μg/plate in the absence and presence of S9-mix, respectively, in the strains TA1535, TA1537 and TA98. The substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of 17 and 164 μg/plate in the absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.
The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

HISTORICAL CONTROL DATA
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 128 – 1530 73 – 1481 58 – 1422 54 – 1239 365 – 1978 250 – 2018
Mean 919 256 802 328 1305 910
SD 172 122 362 154 236 355
n 3215 3122 2777 3187 3202 3216

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2379 93 – 1999 109 - 1968
Mean 907 1308 1073 437
SD 167 386 537 158
n 3231 3179 2923 2987

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 61 8 – 60 61 – 176 60 - 176 10 – 61 9 - 68
Mean 10 11 6 6 16 22 112 108 27 33
SD 3 3 2 3 5 7 18 21 8 9
n 3303 3265 3232 3212 3251 3326 3336 3246 3021 2993

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.
Conclusions:
In an Ames test, performed according to OECD 471 guideline and GLP principles, the substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 164 and 512 μg/plate in the absence and presence of S9-mix, respectively, in the strains TA1535, TA1537 and TA98. The substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test item was tested up to concentrations of 164 μg/plate in the absence of S9-mix in both tester strains and up to 512 and 1600 μg/plate in tester strains TA100 and WP2uvrA, respectively, in the presence of S9-mix. The substance did not precipitate on the plates at any dose level tested.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 17 and 164 μg/plate in the absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 April 2020 - 31 May 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Sex, age and number of blood donors:
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2019): age 25 - 26; AGT: 13.2 - 14.8h
- Whether whole blood or separated lymphocytes were used:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Mitogen used for lymphocytes: Phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 45 - 99%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies).
The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was
1.8% (v/v).
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 24hr exposure; 24hr fixation: 1, 2, 4, 8, 16 and 31 μg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure time, 24hr fixation time: 1, 2, 4, 8, 16 and 31 μg/mL
The following doses were selected for scoring of chromosome aberrations:
Without and with S9-mix, 3hr exposure time, 24hr fixation time: 1, 16 and 31 μg/mL
Second cytogenetic test:
Without S9-mix, 24hr exposure; 24hr fixation: 1, 10, 15, 20, 25 and 31 μg/mL
The following doses were selected for scoring of chromosome aberrations:
Without S9-mix, 24hr exposure; 24hr fixation: 1, 25 and 31 μg/mL
Vehicle / solvent:
- Solvent used: ethanol
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear yellow solution in ethanol. The final concentration of the solvent in the culture medium was 0.5%. Test item concentrations were used within 2 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hr
- Exposure duration/duration of treatment: 3 hr (with and without S9-mix), 24 hr (without S9-mix)
- Harvest time after the end of treatment (sampling/recovery times): 24 hr

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine (0.5 μg/mL medium) during the last 2.5 - 3 h of the culture period.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): One hundred and eighteen to one hundred and fifty metaphase chromosome spreads per culture were examined in duplicate.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).
Evaluation criteria:
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA).
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest precipitating concentration of 31 μg/mL at the 24hr treatment time without S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated at the concentrations of 31 μg/mL.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity was observed at the highest precipitating concentration of 31 μg/mL at the 24hr treatment time without S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database.
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells (see Appendix 3). In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without S9-mix. Cytotoxicity was observed at the highest precipitating concentration of 31 μg/mL at the 24hr treatment time without S9-mix.

- Genotoxicity results (for both cell lines and lymphocytes) : Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
3 hours exposure 24 hours exposure
Gaps included Gaps excluded Gaps included Gaps excluded
-S9 mix +S9 mix -S9 mix +S9 mix -S9 mix -S9 mix
Number of aberrant cells (per 300 cells) 69.0 62.8 67.9 60.5 85.5 83.9
SD 34.3 29.5 34.8 28.6 43.1 43.4
n 68 67 68 67 66 66
Lower Control Limit (95% Control Limits) 2 5 0 5 1 -1
Upper Control Limit (95% Control Limits) 136 121 136 117 170 169
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between October 2016 and October 2019.

- Negative (solvent/vehicle) historical control data:
3 hours exposure 24 hours exposure
Gaps included Gaps excluded Gaps included Gaps excluded
-S9 mix +S9 mix -S9 mix +S9 mix -S9 mix -S9 mix
Number of aberrant cells (per 300 cells) 1.0 0.8 0.8 0.7 0.9 0.7
SD 1.1 1.0 1.0 0.8 1.2 1.1
n 67 67 67 67 66 66
Lower Control Limit (95% Control Limits) -1 -1 -1 -1 -1 -1
Upper Control Limit (95% Control Limits) 3 3 3 2 3 3
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between October 2016 and October 2019.

- Historical Control Data for Numerical Aberrations for in vitro Chromosome Aberration Studies of the Solvent Control:
3 hours exposure 24 hours exposure
Poly Endo Poly Endo
-S9 mix +S9 mix -S9 mix +S9 mix -S9 mix -S9 mix
Number of aberrant cells (per 300 cells) 0.0 0.1 0.0 0.0 0.1 0.0
SD 0.1 0.3 0.2 0.1 0.4 0.2
n 67 67 67 67 66 66
Lower Control Limit (95% Control Limits) 0 0 0 0 -1 0
Upper Control Limit (95% Control Limits) 0 1 0 0 1 0
SD = Standard deviation
n = Number of observations
Poly = polyploidy
Endo = endoreduplication
Distribution historical negative control data from experiments performed between October 2016 and October 2019.

Conclusions:
A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study with the substance was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

The test item precipitated at the concentrations of 31 μg/mL. No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without S9-mix. Cytotoxicity was observed at the highest precipitating concentration of 31 μg/mL at the 24hr treatment time without S9-mix. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that the substance is not clastogenic in human lymphocytes.

Additional information

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 164 and 512 μg/plate in the absence and presence of S9-mix, respectively, in the strains TA1535, TA1537 and TA98. The substance did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test item was tested up to concentrations of 164 μg/plate in the absence of S9-mix in both tester strains and up to 512 and 1600 μg/plate in tester strains TA100 and WP2uvrA, respectively, in the presence of S9-mix. The substance did not precipitate on the plates at any dose level tested.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 17 and 164 μg/plate in the absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

A chromosome aberration study with the substance was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

The test item precipitated at the concentrations of 31 μg/mL. No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without S9-mix. Cytotoxicity was observed at the highest precipitating concentration of 31 μg/mL at the 24hr treatment time without S9-mix. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that the substance is not clastogenic in human lymphocytes.

Justification for classification or non-classification