Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 476, GLP, Read-across, K, rel. 1): non mutagenic.
- Human lymphocytes chromosome aberration test (OECD 473, GLP, Read-across, K, rel. 1): non 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:
From October 05 to November 07, 2016
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:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on 05 July 2016 / signed on 28 October 2016
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was pre-prepared using standardized in-house procedures (outside the confines of this study). The 10% S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 (Pre-Incubation Method):
- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (8.8%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - test item dose levels were selected based on Experiment 1 results in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA100 and TA1535 from 500 μg/plate in Experiment 1.
- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 µg/plate.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Not applicable


COMPARISON WITH HISTORICAL CONTROL DATA:
The individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 dosed in both the absence and presence of S9-mix from 500 µg/plate and to TA98 and TA1537 at 5000 µg/plate (absence of S9-mix only). No toxicity was noted to Escherichia coli strain WP2uvrA in both the absence or presence of S9-mix and TA98 and TA1537 dosed in the presence of S9-mix. Consequently, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 150 µg/plate (TA100, TA98 and TA1537) and 500 µg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100), 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA).

OTHERS:
In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5 μg/plate (TA100 -S9-mix) and in the second mutation test at 15 µg/plate (TA1535 -S9-mix) and 500 µg/plate (WP2uvrA +S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.

- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.
Remarks on result:
other:
Remarks:
Cytotoxicity Experiment 1: +/-S9 mix: TA100 and TA1535 from 500 µg/plate. +S9 mix: TA98 and TA1537 at 5000 µg/plate. Experiment 2: - S9-mix: TA1535 from 50 µg/plate, TA100, TA98 and TA1537 from 150 µg/plate and WP2uvrA from 500 µg/plate. + S9-mix: TA100 from 150 µg/plate, TA1535 and TA1537 from 500 µg/plate and TA98 and WP2uvrA from 1500 µg/plate.

 Table 7.6.1/2. Mutagenic and cytotoxic effect of the test material.

Strain

S9-mix

Test concentration range

(µg/plate)

Lowest mutagenic concentration (µg/plate)

Lowest cytotoxic

concentration (µg/plate)

 

TA100

-

0.5 - 5000

None

150

+

1.5 - 5000

None

150

TA1535

-

0.5 - 5000

None

50

+

1.5 - 5000

None

500

WP2uvrA

-

1.5 - 5000

None

500

+

1.5 - 5000

None

1500

TA98

-

1.5 - 5000

None

150

+

1.5 - 5000

None

1500

TA1537

-

1.5 - 5000

None

150

+

1.5 - 5000

None

500

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

Experiment 1 (Plate Incorporation Method):

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Experiment 2 (Pre-Incubation Method):

- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

 

Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 dosed in both the absence and presence of S9-mix from 500 µg/plate and to TA98 and TA1537 at 5000 µg/plate (absence of S9-mix only). No toxicity was noted to Escherichia coli strain WP2uvrA in both the absence or presence of S9-mix and TA98 and TA1537 dosed in the presence of S9-mix. Consequently, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 150 µg/plate (TA100, TA98 and TA1537) and 500 µg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100), 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 µg/plate.

 

In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5 μg/plate (TA100 -S9 -mix) and in the second mutation test at 15 μg/plate (TA1535 -S9 -mix) and 500 µg/plate (WP2uvrA -S9 -mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls. Further statistically significant increases in revertant colony frequency were also observed in both the first and second mutation tests, however these increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore, the responses are considered false and due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June to 31 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 473 Guideline without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell chromosome aberration assay
Target gene:
None
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
1.8% (v/v) S9-fraction in the exposure medium; S9 fraction prepared from male Sprague-Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (48 h exposure time, 48 h fixation time)
With S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time)

Cytogenetic assay
First cytogenetic assay:
Without & With S9-mix: 5, 50, 75, 100, 125, 150, 175 and 200 μg/mL culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay:
Without S9-mix: 10, 50, 75, 100, 125 and 150 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5, 10, 20, 30, 40, 50, 75 and 100 μg/mL culture medium (48 h exposure time, 48 h fixation time)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: No correction was made for the purity/composition of the test compound. A solubility test was performed to select the appropriate vehicle. The substance did not form a homogeneous suspension in culture medium. Consequently, test substance was dissolved in DMSO of spectroscopic quality. Test substance concentrations were used within 2.5 hours after preparation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
TEST SYSTEM:
Cultured peripheral human lymphocytes were used as test system. 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 2014) are presented below:
Dose range finding study: age 32, AGT = 12.8 h
First cytogenetic assay: age 27, AGT = 12.6 h
Second cytogenetic assay: age 20, AGT = 13.0 h

PREPARATION OF CULTURES:
- Blood samples: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added. All cultures were then incubated in the dark at 37.0 ± 1.0 °C (actual range 35.6 -37.4 °C); humid atmosphere of 80 - 100% (actual range 65 - 91%), containing 5.0 ± 0.5% CO2 in air.

DURATION
- Exposure duration: Dose range finding test and cytogenetic assay: 3 h (±S9); 24 and 48 h (-S9)
- Fixation time (start of exposure up to harvest of cells): 3 h exposure time, 24 h fixation time; 24 h exposure time, 24 h fixation time; 48 h exposure time, 48 h fixation time

SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium).

STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS:
- Dose range finding test: Single culture for test substance and vehicle control
- Main test: Duplicate cultures for test substance, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After arresting the cell division, the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol : acetic acid fixative (3:1 v/v). Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene / pertex and mounted with a coverslip in an automated coverslipper.

NUMBER OF CELLS EVALUATED:
- 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%).
- One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 37 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

OTHERS
- The number of cells with aberrations and the number of aberrations were calculated.
- Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations.
Evaluation criteria:
Equivocal results will be clarified by further testing using modification of experimental conditions.
A test substance 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) Any of the results are outside the 95% control limits of the historical control data range.
A test substance 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) All results are inside the 95% control limits of the negative historical control data range.
In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test substance groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.
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
- Precipitation: At 512 μg/mL, test substance precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
At a concentration of 512 μg/mL test substance precipitated in the culture medium. Therefore, a concentration of 512 μg/mL was used as the highest concentration of test substance.
Without S9-mix (3 h exposure time, 24 h fixation time): 100, 94, 78, 83 and 28% mitotic index at 0, 5.4, 17, 52 and 164 μg/mL, respectively. Cell lysis was observed at 512 μg/mL.
Without S9-mix (24 h exposure time, 24 h fixation time): 100, 100, 94, 68, 4 and 0% mitotic index at 0, 5.4, 17, 52, 164 and 512 μg/mL, respectively
Without S9-mix (48 h exposure time, 48 h fixation time): 100, 83, 57 and 46% mitotic index at 0, 5.4, 17 and 52 μg/mL, respectively. Cell lysis was observed at 164 and 512 μg/mL.
With S9-mix (3 h exposure time, 24 h fixation time): 100, 99, 90, 88 and 47% mitotic index at 0, 5.4, 17, 52 and 164 μg/mL, respectively. Cell lysis was observed at 512 μg/mL.

CYTOGENETIC ASSAY
First cytogenetic assay:
Mitotic index:
Without S9-mix (3 h exposure time, 24 h fixation time): 100, 94, 84, 82, 77, 35, 31 and 19% mitotic index at 0, 5, 50, 75, 100, 125, 150 and 175 μg/mL, respectively. Cell lysis was observed at 200 μg/mL.
With S9-mix (3 h exposure time, 24 h fixation time): 100, 88, 85, 81, 79, 58, 40, 39 and 21% mitotic index at 0, 5, 50, 75, 100, 125, 150, 175 and 200 μg/mL, respectively
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix: 5, 100 and 125 μg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 5, 100 and 150 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:
Mitotic index:
Without S9-mix (24 h exposure time, 24 h fixation time): 100, 86, 74, 66, 39, 25 and 2% mitotic index at 0, 10, 50, 75, 100, 125 and 150 μg/mL, respectively
Without S9-mix (48 h exposure time, 48 h fixation time): 100, 92, 90, 75, 63, 62, 56, 53 and 28% mitotic index at 0, 5, 10, 20, 30, 40, 50, 75 and 100 μg/mL, respectively
At the 48 h exposure time no proper toxicity was reached, 47% toxicity was observed at the dose of 75 μg/mL and 72% toxicity was observed at the dose of 100 μg/mL. Since already narrow dosing steps were chosen, the dose of 75 μg/mL was used to score for chromosome aberrations (3% deviation from the preferred toxicity level).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 75 and 100 μg/mL culture medium (24 h exposure time, 24 h fixation time); 10, 30 and 75 μg/mL culture medium (48 h exposure time, 48 h fixation time).

Both in the absence and presence of S9-mix test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. Although in the second cytogenetic assay at the highest dose of 100 μg/mL at the 24 h exposure time the number of chromosomal aberrations was statistically different compared to the control, the number of aberrations was within the historical data range (7 cells with aberrations per 300 metaphases compared with control limits (+ gaps) -1.79 – 3.51 per 100 metaphases and (- gaps) -1.51 – 2.84 per 100 metaphases) and therefore this increase was considered not biologically relevant. No effects of test substance 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 test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions tested.

HISTORICAL CONTROL DATA
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.
- See table 7.6.1/1 for the historical control data

Table 7.6.1/1: Historical control data

Exposure time

3 h exposure time

 

24 h exposure time

 

48 h exposure time

 

Types

Gaps included

 

Gaps excluded

 

Gaps included

 

Gaps excluded

 

Gaps included

 

Gaps excluded

 

± S9

+ S9-mix

 

- S9-mix

 

+ S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

Historical negative control data for lymphocyte chromosome aberration studies

Mean number of aberrant cells per 100 cells

 

0.86

0.85

0.75

0.77

0.86

0.67

1.05

0.80

SD

 

1.15

1.18

1.06

1.11

1.14

1.06

1.27

1.17

n

226

224

226

224

222

222

220

220

Upper control limit

(95% control limits)

3.65

3.40

3.31

3.06

3.51

2.84

4.28

3.38

Lower control limit

(95% control limits)

-1.93

-1.70

-1.80

-1.52

-1.79

-1.51

-2.18

-1.77

Historical positive control data for lymphocyte chromosome aberration studies

Mean number of aberrant cells per 100 cells

 

27.2

24.6

26.6

24.1

25.8

24.9

28.6

27.7

SD

 

7.13

8.12

6.68

8.06

8.27

8.33

8.65

8.32

n

224

224

224

224

222

222

220

220

Upper control limit

(95% control limits)

43.69

39.47

42.40

38.83

41.82

41.04

45.15

43.66

Lower control limit

(95% control limits)

10.77

9.72

10.72

9.47

9.81

8.84

12.07

11.72

Historical negative control data for numerical aberrations for lymphocyte chromosome aberration studies

Types

Poly

 

 

 

Endo

 

 

 

Poly

 

Endo

 

Poly

 

Endo

 

± S9

+ S9-mix

 

- S9-mix

 

+ S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

Mean number of aberrant cells per 100 cells

 

0.08

0.09

0.01

0.03

0.09

0.01

0.10

0.01

SD

 

0.34

0.32

0.09

0.16

0.35

0.12

0.35

0.12

n

226

224

226

224

222

222

220

220

Upper control limit

(95% control limits)

0.53

0.58

0.06

0.17

0.46

0.09

0.58

0.09

Lower control limit

(95% control limits)

-0.37

-0.40

-0.04

-0.12

-0.29

-0.06

-0.39

-0.06

SD = Standard deviation

n = Number of observations

Poly = polyploidy

Endo = endoreduplication

Distribution historical negative / positive control data from experiments performed between January 2012 and June 2015.

Conclusions:
Based on the results, it is concluded that test substance is not clastogenic in human lymphocytes under the experimental conditions.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to test substance at the following concentrations:

Dose range finding test:

Without S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time)

Cytogenetic assay:

First cytogenetic assay: Without & With S9-mix: 5, 50, 75, 100, 125, 150, 175 and 200 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay: Without S9-mix: 10, 50, 75, 100, 125 and 150 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5, 10, 20, 30, 40, 50, 75 and 100 μg/mL culture medium (48 h exposure time, 48 h fixation time) 

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium).The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was 1.8% (v/v) rat liver S9-fraction.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Based on the mitotic indices, the following dose levels were selected for scoring of chromosome aberrations:

First cytogenetic assay:

without S9-mix: 5, 100 and 125 μg/mL culture medium (3 h exposure time, 24 h fixation time); with S9-mix: 5, 100 and 150 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:

without S9-mix: 10, 75 and 100 μg/mL culture medium (24 h exposure time, 24 h fixation time); 10, 30 and 75 μg/mL culture medium (48 h exposure time, 48 h fixation time).

 

Test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of test substance 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 test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions tested.

 

Based on the results, it is concluded that test substance is not clastogenic in human lymphocytes under the experimental conditions.

This study is considered as acceptable and satisfies the requirement for in vitro cytogenicity in mammalian cells endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October to 07 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 490 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 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)
Qualifier:
according to guideline
Guideline:
other: The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation assay
Target gene:
TK+/- gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Stock cultures of the cells were stored in liquid nitrogen (-196 °C).
- Cell density was preferably kept below 1 x 10^6 cells/mL

MEDIA USED
- Type and identity of media:
Horse serum: Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes; prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (4 % v/v) in the exposure medium; S9 fraction was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Dose range finding test:
3 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, with and without S9 mix
24 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, without S9 mix

Main experiments:
Experiment 1 (3 h treatment):
0.1, 1, 10, 20, 30, 35, 40, 45, 50 and 60 μg/mL, without S9 mix
0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 μg/mL, with S9 mix
Experiment 2 (24 h treatment):
0.1, 1, 5, 10, 20, 30, 40, 50, 60, 75 and 100 μg/mL, without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test item preparation: No correction was made for the purity/composition of the test item. The test item was dissolved in DMSO. The final concentration of the solvent in the exposure medium was 1 % (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;
Exposure medium:
For 3 hour exposure: Cells were exposed to the test item in basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium).
For 24 hour exposure: Cells were exposed to the test item in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).
- Cell density at seeding: Per culture 8 x 10^6 cells (10^6 cells/mL for 3 h treatment) or 5 x 10^6 cells (1.25 x 10^5 cells/mL for 24 h treatment) were used for dose range finding and mutagenicity tests.

DURATION
- Exposure duration: Dose range finding test: 3 h (±-S9-mix); 24 h (- S9-mix); First mutagenicity test: 3 h (±-S9-mix); Second mutagenicity test: 24 h (- S9-mix)
- Expression time (cells in growth medium): 2 days after the treatment period
- Selection time (if incubation with a selection agent): 11 or 12 days
- All incubations were carried out in a humid atmosphere (80 - 100%, actual range 54 – 90%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.3 – 37.3 °C).

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
Dose range finding test: Single culture/dose for test item and vehicle control
Mutagenicity test: Single culture/dose for test item and positive control; duplicate cultures for vehicle control

NUMBER OF CELLS EVALUATED:
- For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non selective medium.
- For determination of the MF a total number of 9.6 x 10^5 cells/concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). In the first experiment in the presence of S9-mix, a total number of 384 wells were used for determination of the mutation frequency in the treatment group of 50 μg/ml instead of 480 as specified in the protocol. The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 2 h, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

DETERMINATION OF CYTOTOXICITY
- Method: Relative Suspension Growth (RSG)

- OTHER:
Determination of the mutant colonies: The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.
Evaluation criteria:
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if:none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
In addition to the criteria stated above, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
Note: The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126
Statistics:
No data
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance was precipitated in the exposure medium at concentrations of 512 μg/mL and above, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL.

DOSE RANGE FINDING TEST:
- In the absence of S9-mix (3 h treatment), the relative suspension growth was 11% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
- In the presence of S9-mix (3 h treatment), the relative suspension growth was 73% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
- In the absence of S9-mix (24 h treatment), the relative suspension growth was 24% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 164 μg/mL and 512 μg/mL after 24 h treatment and no cells survived the 24 h subculture period.

HISTORICAL CONTROL DATA
- The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.
- Positive historical control data: 501-1614 (866 ± 232) MF per 10^6 survivors (without S9-mix, 3 h treatment); 462-1445 (734 ± 196) MF per 10^6 survivors (without S9-mix, 24 h treatment); 593-4356 (1477 ± 678) MF per 10^6 survivors (with S9-mix)
- Negative (solvent/vehicle) historical control data: 50-119 (73 ± 15) MF per 10^6 survivors (without S9-mix, 3 h treatment); 50-170 (74 ± 23) MF per 10^6 survivors (without S9-mix, 24 h treatment); 50-162 (79 ± 22) MF per 10^6 survivors (with S9-mix)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
- In the absence of S9-mix, the dose levels of 0.1 and 1 μg/mL showed no cytotoxicity. Therefore, the dose level of 0.1 μg/mL was not regarded relevant for mutation frequency measurement. The dose level of 60 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
- In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): In the repeat experiment, the following dose range was selected: 0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 μg/mL exposure medium.
- The dose levels of 25 and 65 to 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (25 μg/mL).
- The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 10, 20, 30, 35, 40, 45 and 50 μg/mL exposure medium.
With S9-mix: 0.1, 0.5, 1, 5, 10, 50, 55 and 60 μg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test item concentration was 11% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test item concentration was 14% compared to the total growth of the solvent controls.

Second mutagenicity test:
- The dose levels of 20 and 30 μg/mL showed similar cell growth delay, therefore, the dose level of 20 μg/mL was not regarded relevant for mutation frequency. The dose levels of 75 and 100 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
- The dose levels selected to measure mutation frequencies at the TK-locus were: 0.1, 1, 5, 10, 30, 40, 50 and 60 μg/mL exposure medium.
- The relative total growth of the highest test item was 20% compared to the total growth of the solvent controls.

EVALUATION OF THE MUTAGENICITY
No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 7.6.1/1: Cytotoxic and mutagenic response of the test item in the mouse lymphoma L5178Y test system 

 

Dose (μg/mL)

RSG (%)

CEday2 (%)

RSday2 (%)

RTG (%)

mutation frequency per 106 survivors

Total

small

large

Experiment 1: Without metabolic activation - 3 h treatment

DMSO

100

75

100

100

96

40

52

DMSO

70

60

29

30

1

84

104

143

120

68

25

41

10

74

72

100

74

74

29

43

20

69

79

109

76

95

48

44

30

76

95

131

100

63

35

26

35

61

88

121

74

72

33

37

40

51

107

147

76

53

29

22

45

25

94

130

33

56

30

25

50

10

80

111

11

88

49

36

MMS

63

37

51

32

1037

497

443

Experiment 1: With metabolic activation - 3 h treatment

DMSO

100

105

100

100

81

51

26

DMSO

110

74

43

28

0.1

78

99

92

72

145

84

51

0.5

83

116

108

90

150

94

44

1

84

105

98

83

139

93

38

5

86

107

99

86

161

107

43

10

66

108

101

67

156

118

30

50

52

125

116

60

129

92

29

55

27

97

90

24

133

80

45

60

8

196

182

14

59

29

26

CP

22

12

11

2

3636

2702

673

Experiment 2: Without metabolic activation - 24 h treatment

DMSO

100

97

100

100

75

41

31

DMSO

108

83

57

23

0.1

99

104

101

101

62

40

21

1

93

91

89

82

80

46

30

5

100

84

82

82

55

32

21

10

99

85

83

82

78

46

30

30

71

111

109

77

57

31

24

40

55

105

103

56

57

40

15

50

31

111

109

34

59

39

18

60

18

113

110

20

70

37

30

MMS

78

85

83

65

944

501

270

 

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

Conclusions:
Under the test conditions, test substance is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 490 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test substance at the following concentrations:

Dose range finding test:

3 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, with and without S9 mix

24 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, without S9 mix

Main experiments:

Experiment 1 (3 h treatment): 0.1, 1, 10, 20, 30, 35, 40, 45, 50 and 60 μg/mL, without S9 mix; 0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 μg/mL, with S9 mix

Experiment 2 (24 h treatment): 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 75 and 100 μg/mL, without S9 mix 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 4 % (v/v) rat liver S9 mix.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

Test substance was precipitated in the exposure medium at concentrations of 512 μg/mL and above, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL. In dose range finding test (3 h treatment), the relative suspension growth was 11 and 73% (without and with S9 mix, respectively) at 52 μg/mL compared to the relative suspension growth of the solvent control. In the absence of S9-mix (24 h treatment), the relative suspension growth was 24% at 52 μg/mL. No cell survival was observed at ≥ 164 μg/mL (3 and 24 h treatment).

 

In the first experiment, the relative total growth (RTG) at 50 and 60 μg/mL was 11 and 14% in the absence and presence of S9-mix, respectively. In the second experiment, the relative total growth at 60 μg/mL was 20% compared to the total growth of the solvent controls. No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

 

Under the test conditions, test substance is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for in vitro gene mutation in mammalian cells endpoint.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
- In vitro gene mutation study in mammalian cells
In the MLA performed on the source substance (OECD 476, GLP), none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
- In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance (OECD 473, GLP), no significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the MLA and CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2 and 8.4.3.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
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
- Precipitation: At 512 μg/mL, test substance precipitated in the culture medium.

RANGE-FINDING/SCREENING STUDIES:
At a concentration of 512 μg/mL test substance precipitated in the culture medium. Therefore, a concentration of 512 μg/mL was used as the highest concentration of test substance.
Without S9-mix (3 h exposure time, 24 h fixation time): 100, 94, 78, 83 and 28% mitotic index at 0, 5.4, 17, 52 and 164 μg/mL, respectively. Cell lysis was observed at 512 μg/mL.
Without S9-mix (24 h exposure time, 24 h fixation time): 100, 100, 94, 68, 4 and 0% mitotic index at 0, 5.4, 17, 52, 164 and 512 μg/mL, respectively
Without S9-mix (48 h exposure time, 48 h fixation time): 100, 83, 57 and 46% mitotic index at 0, 5.4, 17 and 52 μg/mL, respectively. Cell lysis was observed at 164 and 512 μg/mL.
With S9-mix (3 h exposure time, 24 h fixation time): 100, 99, 90, 88 and 47% mitotic index at 0, 5.4, 17, 52 and 164 μg/mL, respectively. Cell lysis was observed at 512 μg/mL.

CYTOGENETIC ASSAY
First cytogenetic assay:
Mitotic index:
Without S9-mix (3 h exposure time, 24 h fixation time): 100, 94, 84, 82, 77, 35, 31 and 19% mitotic index at 0, 5, 50, 75, 100, 125, 150 and 175 μg/mL, respectively. Cell lysis was observed at 200 μg/mL.
With S9-mix (3 h exposure time, 24 h fixation time): 100, 88, 85, 81, 79, 58, 40, 39 and 21% mitotic index at 0, 5, 50, 75, 100, 125, 150, 175 and 200 μg/mL, respectively
The following dose levels were selected for scoring of chromosome aberrations:
Without S9-mix: 5, 100 and 125 μg/mL culture medium (3 h exposure time, 24 h fixation time).
With S9-mix: 5, 100 and 150 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:
Mitotic index:
Without S9-mix (24 h exposure time, 24 h fixation time): 100, 86, 74, 66, 39, 25 and 2% mitotic index at 0, 10, 50, 75, 100, 125 and 150 μg/mL, respectively
Without S9-mix (48 h exposure time, 48 h fixation time): 100, 92, 90, 75, 63, 62, 56, 53 and 28% mitotic index at 0, 5, 10, 20, 30, 40, 50, 75 and 100 μg/mL, respectively
At the 48 h exposure time no proper toxicity was reached, 47% toxicity was observed at the dose of 75 μg/mL and 72% toxicity was observed at the dose of 100 μg/mL. Since already narrow dosing steps were chosen, the dose of 75 μg/mL was used to score for chromosome aberrations (3% deviation from the preferred toxicity level).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 75 and 100 μg/mL culture medium (24 h exposure time, 24 h fixation time); 10, 30 and 75 μg/mL culture medium (48 h exposure time, 48 h fixation time).

Both in the absence and presence of S9-mix test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. Although in the second cytogenetic assay at the highest dose of 100 μg/mL at the 24 h exposure time the number of chromosomal aberrations was statistically different compared to the control, the number of aberrations was within the historical data range (7 cells with aberrations per 300 metaphases compared with control limits (+ gaps) -1.79 – 3.51 per 100 metaphases and (- gaps) -1.51 – 2.84 per 100 metaphases) and therefore this increase was considered not biologically relevant. No effects of test substance 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 test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions tested.

HISTORICAL CONTROL DATA
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range.
- See table 7.6.1/1 for the historical control data

Table 7.6.1/1: Historical control data

Exposure time

3 h exposure time

 

24 h exposure time

 

48 h exposure time

 

Types

Gaps included

 

Gaps excluded

 

Gaps included

 

Gaps excluded

 

Gaps included

 

Gaps excluded

 

± S9

+ S9-mix

 

- S9-mix

 

+ S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

Historical negative control data for lymphocyte chromosome aberration studies

Mean number of aberrant cells per 100 cells

 

0.86

0.85

0.75

0.77

0.86

0.67

1.05

0.80

SD

 

1.15

1.18

1.06

1.11

1.14

1.06

1.27

1.17

n

226

224

226

224

222

222

220

220

Upper control limit

(95% control limits)

3.65

3.40

3.31

3.06

3.51

2.84

4.28

3.38

Lower control limit

(95% control limits)

-1.93

-1.70

-1.80

-1.52

-1.79

-1.51

-2.18

-1.77

Historical positive control data for lymphocyte chromosome aberration studies

Mean number of aberrant cells per 100 cells

 

27.2

24.6

26.6

24.1

25.8

24.9

28.6

27.7

SD

 

7.13

8.12

6.68

8.06

8.27

8.33

8.65

8.32

n

224

224

224

224

222

222

220

220

Upper control limit

(95% control limits)

43.69

39.47

42.40

38.83

41.82

41.04

45.15

43.66

Lower control limit

(95% control limits)

10.77

9.72

10.72

9.47

9.81

8.84

12.07

11.72

Historical negative control data for numerical aberrations for lymphocyte chromosome aberration studies

Types

Poly

 

 

 

Endo

 

 

 

Poly

 

Endo

 

Poly

 

Endo

 

± S9

+ S9-mix

 

- S9-mix

 

+ S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

- S9-mix

 

Mean number of aberrant cells per 100 cells

 

0.08

0.09

0.01

0.03

0.09

0.01

0.10

0.01

SD

 

0.34

0.32

0.09

0.16

0.35

0.12

0.35

0.12

n

226

224

226

224

222

222

220

220

Upper control limit

(95% control limits)

0.53

0.58

0.06

0.17

0.46

0.09

0.58

0.09

Lower control limit

(95% control limits)

-0.37

-0.40

-0.04

-0.12

-0.29

-0.06

-0.39

-0.06

SD = Standard deviation

n = Number of observations

Poly = polyploidy

Endo = endoreduplication

Distribution historical negative / positive control data from experiments performed between January 2012 and June 2015.

Conclusions:
Based on the results on the source substance, it is concluded that the target substance is not clastogenic in human lymphocytes under the experimental conditions.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to the source substance at the following concentrations:

Dose range finding test:

Without S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5.4, 17, 52, 164 and 512 μg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix: 5.4, 17, 52, 164 and 512 μg/mL culture medium (3 h exposure time, 24 h fixation time)

Cytogenetic assay:

First cytogenetic assay: Without & With S9-mix: 5, 50, 75, 100, 125, 150, 175 and 200 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay: Without S9-mix: 10, 50, 75, 100, 125 and 150 μg/mL culture medium (24 h exposure time, 24 h fixation time); 5, 10, 20, 30, 40, 50, 75 and 100 μg/mL culture medium (48 h exposure time, 48 h fixation time) 

During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium).The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Metabolic activation system used in this test was 1.8% (v/v) rat liver S9-fraction.

 

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Based on the mitotic indices, the following dose levels were selected for scoring of chromosome aberrations:

First cytogenetic assay:

without S9-mix: 5, 100 and 125 μg/mL culture medium (3 h exposure time, 24 h fixation time); with S9-mix: 5, 100 and 150 μg/mL culture medium (3 h exposure time, 24 h fixation time).

Second cytogenetic assay:

without S9-mix: 10, 75 and 100 μg/mL culture medium (24 h exposure time, 24 h fixation time); 10, 30 and 75 μg/mL culture medium (48 h exposure time, 48 h fixation time).

 

Test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No effects of test substance 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 test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions tested.

 

Based on the results on the source substance, it is concluded that the target substance is not clastogenic in human lymphocytes under the experimental conditions.

This study is considered as acceptable and satisfies the requirement for in vitro cytogenicity in mammalian cells endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Further information is included as attachment to the Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, (eco)toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and source substances are structurally related, in that both are 1-[(x,x)-dimethyl-1-cyclohexen-1-yl]-pent-4-en-1-one, which can exist as alpha (1-[(5,5)-dimethyl...) or beta (1-[(3,3)-dimethyl...) forms, meaning the position of the double bond in the hexane cycle differs.
The target substance is the isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one).
The source substance is a mixture of isomer alpha (1-(5,5-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present as the major constituent between 60 and 75% in the mixture, and corresponding to the target (mono-constituent) substance; and isomer beta (1-(3,3-Dimethyl-1-cyclohexen-1-yl)-4-penten-1-one), present between 25 and 35%.

3. ANALOGUE APPROACH JUSTIFICATION
- In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
- In vitro gene mutation study in mammalian cells
In the MLA performed on the source substance (OECD 476, GLP), none of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
- In vitro cytogenicity study in mammalian cells
In the CAT performed on the source substance (OECD 473, GLP), no significant increases in the frequency of cells with aberrations was induced. The test material used represents the source substance as described in the hypothesis in terms of purity and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the result of the MLA and CAT conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.2 and 8.4.3.

4. DATA MATRIX
See Iuclid section13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance was precipitated in the exposure medium at concentrations of 512 μg/mL and above, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL.

DOSE RANGE FINDING TEST:
- In the absence of S9-mix (3 h treatment), the relative suspension growth was 11% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
- In the presence of S9-mix (3 h treatment), the relative suspension growth was 73% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test item concentrations of 164 μg/mL and above.
- In the absence of S9-mix (24 h treatment), the relative suspension growth was 24% at the test item concentration of 52 μg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 164 μg/mL and 512 μg/mL after 24 h treatment and no cells survived the 24 h subculture period.

HISTORICAL CONTROL DATA
- The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.
- Positive historical control data: 501-1614 (866 ± 232) MF per 10^6 survivors (without S9-mix, 3 h treatment); 462-1445 (734 ± 196) MF per 10^6 survivors (without S9-mix, 24 h treatment); 593-4356 (1477 ± 678) MF per 10^6 survivors (with S9-mix)
- Negative (solvent/vehicle) historical control data: 50-119 (73 ± 15) MF per 10^6 survivors (without S9-mix, 3 h treatment); 50-170 (74 ± 23) MF per 10^6 survivors (without S9-mix, 24 h treatment); 50-162 (79 ± 22) MF per 10^6 survivors (with S9-mix)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First mutagenicity test:
- In the absence of S9-mix, the dose levels of 0.1 and 1 μg/mL showed no cytotoxicity. Therefore, the dose level of 0.1 μg/mL was not regarded relevant for mutation frequency measurement. The dose level of 60 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing.
- In the presence of S9-mix, too many dose levels showed severe cytotoxicity, this part of the experiment was repeated (experiment 1A): In the repeat experiment, the following dose range was selected: 0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 μg/mL exposure medium.
- The dose levels of 25 and 65 to 80 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (25 μg/mL).
- The dose levels selected to measure mutation frequencies at the TK-locus were:
Without S9-mix: 1, 10, 20, 30, 35, 40, 45 and 50 μg/mL exposure medium.
With S9-mix: 0.1, 0.5, 1, 5, 10, 50, 55 and 60 μg/mL exposure medium.
- In the absence of S9-mix, the relative total growth of the highest test item concentration was 11% compared to the total growth of the solvent controls.
- In the presence of S9-mix, the relative total growth of the highest test item concentration was 14% compared to the total growth of the solvent controls.

Second mutagenicity test:
- The dose levels of 20 and 30 μg/mL showed similar cell growth delay, therefore, the dose level of 20 μg/mL was not regarded relevant for mutation frequency. The dose levels of 75 and 100 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.
- The dose levels selected to measure mutation frequencies at the TK-locus were: 0.1, 1, 5, 10, 30, 40, 50 and 60 μg/mL exposure medium.
- The relative total growth of the highest test item was 20% compared to the total growth of the solvent controls.

EVALUATION OF THE MUTAGENICITY
No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 7.6.1/1: Cytotoxic and mutagenic response of the test item in the mouse lymphoma L5178Y test system 

 

Dose (μg/mL)

RSG (%)

CEday2 (%)

RSday2 (%)

RTG (%)

mutation frequency per 106 survivors

Total

small

large

Experiment 1: Without metabolic activation - 3 h treatment

DMSO

100

75

100

100

96

40

52

DMSO

70

60

29

30

1

84

104

143

120

68

25

41

10

74

72

100

74

74

29

43

20

69

79

109

76

95

48

44

30

76

95

131

100

63

35

26

35

61

88

121

74

72

33

37

40

51

107

147

76

53

29

22

45

25

94

130

33

56

30

25

50

10

80

111

11

88

49

36

MMS

63

37

51

32

1037

497

443

Experiment 1: With metabolic activation - 3 h treatment

DMSO

100

105

100

100

81

51

26

DMSO

110

74

43

28

0.1

78

99

92

72

145

84

51

0.5

83

116

108

90

150

94

44

1

84

105

98

83

139

93

38

5

86

107

99

86

161

107

43

10

66

108

101

67

156

118

30

50

52

125

116

60

129

92

29

55

27

97

90

24

133

80

45

60

8

196

182

14

59

29

26

CP

22

12

11

2

3636

2702

673

Experiment 2: Without metabolic activation - 24 h treatment

DMSO

100

97

100

100

75

41

31

DMSO

108

83

57

23

0.1

99

104

101

101

62

40

21

1

93

91

89

82

80

46

30

5

100

84

82

82

55

32

21

10

99

85

83

82

78

46

30

30

71

111

109

77

57

31

24

40

55

105

103

56

57

40

15

50

31

111

109

34

59

39

18

60

18

113

110

20

70

37

30

MMS

78

85

83

65

944

501

270

 

RSG = Relative Suspension Growth; CE = Cloning Efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = DMSO; MMS = Methylmethanesulfonate; CP = Cyclophosphamide

Conclusions:
Based on the results on the source substance, the target substance is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 490 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to the source substance at the following concentrations:

Dose range finding test:

3 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, with and without S9 mix

24 h treatment: 5.4, 17, 52, 164 and 512 μg/mL, without S9 mix

Main experiments:

Experiment 1 (3 h treatment): 0.1, 1, 10, 20, 30, 35, 40, 45, 50 and 60 μg/mL, without S9 mix; 0.1, 0.5, 1, 5, 10, 25, 50, 55, 60, 65, 70 and 80 μg/mL, with S9 mix

Experiment 2 (24 h treatment): 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 75 and 100 μg/mL, without S9 mix 

Vehicle and positive control groups were also included in each mutation test. Metabolic activation system used in this test was 4 % (v/v) rat liver S9 mix.

 

The spontaneous mutation frequencies in the solvent-treated control cultures were within the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay. Mutation frequencies in cultures treated with positive control chemicals were within the acceptability criteria of this assay. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

Test substance was precipitated in the exposure medium at concentrations of 512 μg/mL and above, the concentration used as the highest test item concentration for the dose range finding test was 512 μg/mL. In dose range finding test (3 h treatment), the relative suspension growth was 11 and 73% (without and with S9 mix, respectively) at 52 μg/mL compared to the relative suspension growth of the solvent control. In the absence of S9-mix (24 h treatment), the relative suspension growth was 24% at 52 μg/mL. No cell survival was observed at ≥ 164 μg/mL (3 and 24 h treatment).

 

In the first experiment, the relative total growth (RTG) at 50 and 60 μg/mL was 11 and 14% in the absence and presence of S9-mix, respectively. In the second experiment, the relative total growth at 60 μg/mL was 20% compared to the total growth of the solvent controls. No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

 

Based on the results on the source substance, the target substance is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

This study is considered as acceptable and satisfies the requirement for in vitro gene mutation in mammalian cells endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°

Substance tested

Test / Guideline

Reliability

Focus

Strains tested

Test concentration

Statement

1

 

Envigo, 2017

Target substance

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100

E.coli WP2 uvrA

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

WIL

Research Europe BV, 2015

Source substance

L5178YTK+/-/MLA test (OECD 476)

K, rel. 1

Gene mutation

L5178Y tk+/-(3.7.2C) mouse lymphoma cells

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

3

 

WIL Research Europe BC, 2015

Source substance

HL/CAT

(OECD 473)

K, rel.1

Chromosomal aberration

Human lymphocyte

Up to cytotoxic concentration

-S9 : non clastogenic

+S9 : non clastogenic

Gene mutation Assays (Tests n° 1 -2):

- A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD test guideline No 471 with the substance (See Table 1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria under the test condition whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The target substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammal cells using an in vitro gene mutation assay in L5178Y tk+/-(3.7.2C) mouse lymphoma cells (L5178Y TK+/- /MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit with the target substance (see IUCLID section 13 for read-across justification), either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the source and the target substances are considered as negative for inducing gene mutations at the TK locus in L5178Y mouse lymphoma cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the target substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the test material was determined with the source substance (see IUCLID section 13 for read-across justification) using an in vitro chromosome aberration test in Human lymphocytes, which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured Human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the supporting substance, either with or without metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments. The source substance does not induce structural aberrations in the chromosomes of Human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. Therefore, both the source and the target substances are considered as negative for inducing chromosomal mutations in Human lymphocytes in vitro under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).