1-(3,5-Dichloro-4-fluoro-phenyl)-2,2,2-trifluoro-ethanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non-mutagenic, S. typhimurium TA1537, TA1535, TA98, TA100 and E. coli WP2 and WP2 uvra with and without S9, Ames test, OECD TG 471 (Chang 2020)

Clastogenic/aneugenic, human lymphocytes, in-vitro micronucleus assay, OECD TG 487 (Naumann 2021)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively. The S. typhimurium and E. coli strains are constructed to differentiate between base pair (TA1535, TA100, WP2 uvrA (pKM101), and WP2 (pKM101)) and frameshift (TA1537, TA98) mutations.

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Wistar rats (RjHan:WI; weight approx. 220 – 320 g, Janvier Labs, 53941 Saint-Berthevin Cedex, France) induced by peroral administration of 80 mg/kg b.w. phenobarbital (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) and by peroral administrations of ß-naphthoflavone (Acros Organics, 2440 Geel, Belgium) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene. The protein concentration in the S9 preparation was 30.3 mg/mL (lot no. 281119K, recertified 03 June 2020) in the pre-experiment / Experiment I and 33.0 mg/mL (lot no. 030920K) in Experiments Ia and II.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment Ia: 1, 3, 10, 33, 100, 333 and 1000 µg/plate
Experiment II: 0.3, 1, 3, 10, 33, 100, 333 and 1000 µg/plate
Significant cytotoxicity was observed in experiment I. Furthermore, precipitation occurred at 2500 and 5000 µg/plate in the presence of S9 mix in experiment I.

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Remarks:

Sodium azide without S9 for TA1535 and TA100; 4-nitro-o-phenylene-diamine without S9 for TA1537, TA98; methyl methane sulfonate without S9 for WP2, 2-aminoanthracene with S9 for all strains

Details on test system and experimental conditions:

For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Evaluation criteria:

A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above the spontaneous mutation rate of the corresponding solvent control is observed. A concentration dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration. An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

None

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

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

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Since strong cytotoxic effects were observed in Experiment I eight concentrations were tested in Experiment II. 1000 µg/plate was chosen as the maximal concentration in Experiment II. The test item precipitated in the overlay agar in the test tubes in Experiment I at 5000 µg/plate in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment I from 2500 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording. Based on a lower top concentration of 1000 µg/plate no precipitation of the test substance occurred in the overlay agar in Experiments Ia and II neither in the test tubes nor on the incubated agar plates. No substantial increase in revertant colony numbers in any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies consistent with the laboratory’s historical control data demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Conclusions:

The substance did not induce gene mutations by base pair changes or frameshifts in the genome of the bacterial S. typhimurium and E. coli strains used and was found to be non-mutagenic.

Executive summary:

The potential of the substance to induce gene mutations in the plate incorporation test (Experiments I and Ia) and the pre-incubation test (Experiment II) using the Salmonella typhimurium (S. typhimurium) strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli (E. coli) strains WP2 uvrA (pKM101) and WP2 (pKM101) was studied under GLP to OECD TG 471. The concentrations were 3 to 5000 µg/plate in Experiment I, 1 to 1000 µg/plate in Experiment Ia and 0.3 to 1000 µg/plate in Experiment II. The plates incubated with the test item showed reduced background growth in all strains used with and without S9 mix. Cytotoxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used with and without S9 mix. No relevant increase in revertant colony numbers of any of the six tester strains was observed following treatment with the substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no observed tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls, which showed a distinct increase of induced revertant colonies consistent with the laboratory’s historical control data demonstrated the sensitivity of the test system and the efficacy of the S9 mix. Each batch of S9 was also tested with 2 pro-mutagens, benzo(a)pyrene and 2-aminoanthracene.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable: The occurrence of micronuclei in interphase cells provides an indirect, but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and rarely occur spontaneously, but are inducible by clastogens or agents affecting the spindle apparatus (Countryman and Heddle, 1976; Obe and Beek, 1982, Rosefort et al, 2004).

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Obtained on the days of culture initiation from healthy, non-smoking donors. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.

Cytokinesis block (if used):

Micronuclei should only be evaluated in cells that have completed mitosis during exposure to the test substance or during the post-exposure period and thus a cytokinesis blocker, cytochalasin B, is added to the cell culture to ensure that there are binucleated cells to be evaluated for micronuclei (Rosefort et al, 2004).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Wistar rats (RjHan:WI; Janvier Labs, 53941 Saint-Berthevin Cedex, France) induced by peroral administration of 80 mg/kg b.w. phenobarbital (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) and by peroral administrations of ß-naphthoflavone (Acros Organics, 2440 Geel, Belgium) each, on three consecutive days. The livers were prepared 24 h after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week.
Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test (Ames et al, 1975). An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). The protein concentration of the S9 preparation was 31.7 mg/mL (Lot no. 200220).

Test concentrations with justification for top dose:

Test substance concentrations ranging from 13.0 μg/mL to 2000 μg/mL (with and without S9 mix) were chosen for evaluation of cytotoxicity in the pre-experiment. The highest concentration was chosen with regard to the substance's molecular weight. Considering the cytotoxicity data of the pre-test, 150 μg/mL (with and without S9 mix) was chosen as top treatment concentration for Experiment I.
Experiment I, without S9: 8.8, 15.4, 26.9, 35.0 µg/mL
Experiment I, with S9: 5.0, 8.8, 15.4, 35.0 µg/mL

Vehicle / solvent:

Dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mytomycin C without S9, cyclophosphamide with S9

Details on test system and experimental conditions:

Preliminary Cytotoxicity Assay: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 h (with and without S9 mix). The preparation interval was 40 h after start of the exposure.
Details of slide preparation
Pulse exposure: About 48 h after seeding, 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation S9 mix (50 µL/mL culture medium) was added. After 4 h the cells were spun down by gentle centrifugation for 5 minutes. The supernatant was discarded and the cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4). The washing procedure was repeated once as described. The cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period. After this period Cytochalasin B (4 µg/mL) was added and the cells were cultured another approximately 20 hours until preparation (Clare et al, 2006, Lorge et al, 2006).
Preparation and analysis of cells: The cultures were harvested by centrifugation 40 h after beginning of treatment. The cells were washed and fixated. The slides were added to a microscope slide and stained with Giemsa. Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in binucleated cells showing a clearly visible cytoplasm area. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. The frequency of micronucleated cells was reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture. Cytotoxicity is expressed as cytostasis, calculating the CBPI, and used therefore as a cut off criteria. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Evaluation criteria:

The percentages of micronuclei in binucleate cells were calculated for each treatment scored. The data have been interpreted as follows:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase when assessed by a trend test
- The results in all evaluated test substance concentrations should be within the 95% control limits of the laboratory’s historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test substance concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition when assessed by a trend test
- The results are outside the range of the 95% control limit of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

Statistical significance was confirmed by the Chi square test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression test was performed using a validated test script of "R", to assess a possible concentration dependent increase of micronucleus frequency. The number of micronucleated cells obtained for the groups treated with the test substance was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance were considered together.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the pre-test in the absence of S9 mix, cytotoxicity was observed at 22.7 μg/mL and above. In the presence of S9 mix, cytotoxicity was observed at 39.8 μg/mL and above. Phase separation of the test substance was observed at the end of treatment at 213 μg/mL and above in the presence of S9 mix. The experimental part without S9 mix was repeated due to strong cytotoxicity and no sufficient evaluable concentrations available. The experimental part with S9 mix was repeated because the solvent control was invalid (exceeded the historical control data range).
Considering the cytotoxicity data of the pre-test, 150 μg/mL (with and without S9 mix) was chosen as top treatment concentration for Experiment I.
Phase separation was observed in Experiment I in the presence of S9 mix at 150 μg/mL at the end of treatment. In the absence of S9 mix neither precipitation nor phase separation was observed at the end of treatment.
No relevant influence of the test substance on the osmolarity and pH was observed.
In the main experiment in the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the absence of S9 mix after treatment with the three highest evaluated concentrations (15.4, 26.9 and 35.0 μg/mL), the values of 0.60, 1.95 and 1.10 % micronucleated cells were statistically significantly increased with respect to the concurrent control. The two highest evaluated concentrations (26.9 and 35.0 μg/mL) clearly exceeded the 95 % control limit of the historical control data (0.00 – 1.04 % micronucleated cells). The value of 1.95 % micronucleated cells after treatment with 26.9 μg/mL of the test substance also exceeded the overall min-max range of the historical control data (0.05 – 1.20 % micronucleated cells). No concentration-dependency, tested via trend test was observed. However, the experiment was concluded to be positive.
In the presence of S9 mix, no statistical significance was observed but all values (1.20, 1.60, 1.65 and 1.45 % micronucleated cells) after treatment with 5.0, 8.8, 15.4 and 35.0 μg/mL of the test substance exceeded the 95 % control limit of the historical control data (0.00 – 1.03 % micronucleated cells). The values of the three highest tested concentrations exceeded the overall min-max range of the historical control data (0.05 – 1.25 % micronucleated cells). Again, no concentration-dependency, tested via trend test was observed. However, the experiment was concluded to be positive.
MMC (0.8 μg/mL) or CPA (15.0 μg/mL) were used as appropriate positive control chemicals and showed statistically significant increases in binucleated cells with micronuclei demonstrating the correct performance of the assay.

Remarks on result:

other: Result in experiments with and without S9 considered to be positive.

Summary of results for 4 hour exposure without S9 mix

Experiment	Incubation interval	Concentration in μg/mL	Proliferation index CBPI	Cytostatis in %	Micro-nucleated cells in %	95% confidence interal in %
I	40 hours	DMSO 0.5%	1.71		0.15	0.0 - 1.04
		0.8 μg/mL MMC	1.29	59.2	10.20 S
		8.8	1.66	7.7	0.45
		15.4	1.64	9.4	0.60 S
		26.9	1.39	44.8	1.95 S
		35.0	1.29	58.9	1.10 S

S = The number of micronucleated cells was statistically significantly higher than corresponding control values

Trend test: p-value = 0.114

Summary of results for 4 hour exposure with S9 mix

Experiment	Incubation interval	Concentration in μg/mL	Proliferation index CBPI	Cytostatis in %	Micro-nucleated cells in %	95% confidence interal in %
I	40 hours	DMSO 0.5%	1.61		0.95	0.0 - 1.03
		15 μg/mL CPA	1.26	57.6	4.65 S
		5.0	1.62	n.c.	1.20
		8.8	1.54	11.0	1.60
		15.4	1.40	35.2	1.65
		35.0	1.27	56.6	1.45

S = The number of micronucleated cells was statistically significantly higher than corresponding control values

Trend test: p-value = 0.369

Conclusions:

The substance was found to induce micronuclei in the in vitro test with human lymphocytes and was considered to be genotoxic when tested up to cytotoxic concentrations.

Executive summary:

The test substance, dissolved in DMSO, was assessed under GLP for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments performed to OECD TG 487. In each experimental group, two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The highest applied concentration in this study (2000 µg/mL of the test substance) was chosen with regard to the molecular weight of the test substance. In the absence and presence of S9 mix, clear cytotoxicity was observed at the highest evaluated concentration. In the absence of S9 mix after treatment with the three highest evaluated concentrations (15.4, 26.9 and 35.0 µg/mL), the values of 0.60, 1.95 and 1.10% micronucleated cells were statistically significantly increased with respect to the concurrent control. The two highest evaluated concentrations (26.9 and 35.0 µg/mL) clearly exceeded the 95% control limit of the historical control data (0.00 – 1.04% micronucleated cells). The value of 1.95% micronucleated cells after treatment with 26.9 µg/mL of the test substance also exceeded the overall min-max range of the historical control data (0.05 – 1.20% micronucleated cells). No concentration-dependency, tested via trend test was observed. However, the experiment was concluded to be positive. In the presence of S9 mix, no statistical significance was observed but all values (1.20, 1.60, 1.65 and 1.45% micronucleated cells) after treatment with 5.0, 8.8, 15.4 and 35.0 µg/mL of the test substance exceeded the 95% control limit of the historical control data (0.00 – 1.03% micronucleated cells). The values of the three highest tested concentrations exceeded the overall min-max range of the historical control data (0.05 – 1.25% micronucleated cells). Again, no concentration-dependency, tested via trend test was observed. However, the experiment was concluded to be positive. Appropriate mutagens were used as positive controls. They induced statistically significant increases in binucleated cells with micronuclei demonstrating the correct performance of the assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Non-genotoxic (not clastogenic, not aneugenic), mouse bone marrow erythrocytes, in-vivo micronucleus assay, OECD TG 474 (Dony 2021)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start: 02.02.2021, Experimental completion: 18 March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigations, which may be helpful in the interpretation of results from the micronucleus test.

Sex:

male

Details on test animals or test system and environmental conditions:

Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
Age: 6 to 10 weeks
Acclimation: 5 days
Body weight: 34.8 (31.2 to 37.2) g at start, 35.6 (32.2 to 38.8) g at end of experiment
Housing: individually in Macrolon Type II/III cage with wire mesh top, soft wood bedding
Feed: pellet standard diet ad libitum
Water: tap water ad libitum
Temperature: 22 ± 2°C
Humidity: 45 to 65%
Ventilation: at least 8 air changes per hour
Light: 12 hours light to 12 hours darkness cycles

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Animals received the oral dose by gavage, using a stainless steel feeding needle with rounded tip (1.2 Gauge) and disposable syringe at a dose volume of 10 mL/kg bw.

Duration of treatment / exposure:

Sampling of bone marrow after 24 and 48 hours

Frequency of treatment:

Single oral dose by gavage

Post exposure period:

Animals were sacrificed 24 and 48 hours after oral administration of the test substance or control substances.

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Preliminary study: two male and two female mice
Main study: 6 males per dose, 5 males for negative and positive controls

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA), administered at 40 mg/kg bw in distilled water, administered in a volume of 10 mL/kg bw

Tissues and cell types examined:

Samples were prepared from bone marrow cells obtained from the femora of test animals. Per animal 6000 polychromatic erythrocytes (PCE) were analysed for micronuclei.

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum using a disposable syringe. The cell suspension was centrifuged at 1500 rpm (3900 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Evaluation criteria:

A test substance is classified as positive in the assay if
a) at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
A test item that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying the above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure.

Statistics:

Statistical methods (nonparametric Mann-Whitney test, linear regression analysis) were used as an aid in evaluating the results.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

One animal dosed at 125 mg/kg bw showed piloerection, partially closed eyes and decreased activity in the first hour following oral administration. All animals dosed at 500 mg/kg bw exhibited signs of acute toxicity. All treated animals showed piloerection for up to 4 hours after dosing. A smaller number of animals treated with 500 mg/kg bw showed partially closed eyes, hunched posture, decreased activity and prostation during the first hour following administration.

Table: summary of micronucleus test results

Test group	Dose in mg/kg bw	Sampling time (h)	Mean MN/4000 PCE	SD MN/4000 PCE	Mean % MN	Min	Max	Ratio PCE/total erythroc.	% ratio vehicle
Vehicle control	0	24	3.2	1.2	0.08	2	5	0.582	100.00
Test item	125	24	3.1	1.4	0.08	2	5	0.579	99.48
Test item	250	24	5.0	1.6	0.13	3	7	0.601	103.26
Test item	500	24	5.2	2.0	0.13	2	8	0.613	105.33
Positive control	40	24	114.1	41.4	2.85	69	167	0.643	110.48
Vehicle control	0	48	3.2	0.7	0.08	2	4	0.609	100.00
Test item	500	48	6.0	2.5	0.15	3	9	0.575	94.42

Conclusions:

The substance did not induce micronuclei in polychromatic erythrocytes of mice in this in vivo bone marrow micronucleus assay. It is considered to be non-genotoxic (non-clastogenic and non-aneugenic).

Executive summary:

The genotoxicity potential of the substance was studied under GLP in an in vivo bone marrow microncleus assay in the mouse to OECD TG 474 (2016). The test substance was dissolved in corn oil and administered to male NMRI mice (aged 6 to 10 weeks) by oral gavage. A single dose of 125, 250 and 500 mg/kg bw was administered orally to six animals per group at a volume of 10 mL/kg bw. The highest dose was estimated to be a suitable maximum tolerated dose based on two pre-experiments with two male and two female mice. Clinical signs observed were indicative of systemic exposure to the test substance. The bone marrow of treated animals was collected 24 hours (all dose groups) and 48 hours (500 mg/kg bw) after oral administration. Groups of five animals received the vehicle without test substance (negative vehicle control) and were sacrificed after 24 and 48 hours. Another group of five animals received cyclophosphamide at a single oral dose of 40 mg/kg in distilled water (volume of 10 mL/kg bw) as the positive control substance. Bone marrow was obtained from the femora of test animals and smears on slides were air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample. The slides were evaluated for the occurrence of micronuclei. Per animal at least 6000 polychromatic erythrocytes (PCEs) were scored for micronuclei. After treatment with the test substance the number of PCEs per total erythrocytes was not substantially decreased as compared to the mean value of PCEs per total erythrocytes of the vehicle control, shwowing that the substance did not exert any significant cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls, there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance at any dose level used. For all treatment groups the mean values of micronuclei observed after treatment with the substance were below or near the corresponding vehicle control value and well within the the 95% control limit of the historical vehicle control data. Treatment with the positive control substance produced a substantial increase of induced micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies

The genotoxicity of the substance was studied under GLP in two valid and reliable in vitro tests. In an Ames test conducted to OECD TG 471, there was no relevant increase in revertant colony numbers of any of the six S. typhimurium and E. coli tester strains at any concentration of the test substance (including concentrations causing significant cytotoxicity) with or without S9 mix. The substance was therefore considered to be non-mutagenic.

In an in vitro micronucleus test performed to OECD TG 487 with human lymphocytes, two independent experiments with two parallel cultures were analysed. Per culture 1000 binucleated cells were evaluated for clastogenic damage. Clear cytotoxicity was observed at the highest evaluated concentration. A statistically significant increase in the number of micronucleated cells was observed at the three highest test concentrations without S9 mix. The numbers at the two highest concentrations (26.9 and 35.0 µg/mL) clearly exceeded the 95% control limit of the historical control data and the number of micronucleated cells after treatment with 26.9 µg/mL also exceeded the overall min-max range of the historical control data. The experiment was concluded to be positive, although no concentration-dependency was seen. In the presence of S9 mix, the numbers of micronucleated cells exposed to 5.0, 8.8, 15.4 and 35.0 µg/mL exceeded the 95% control limit of the historical control data, but without statistical significance. The values of the three highest test concentrations exceeded the overall min-max range of the historical control data, but no concentration-dependency was observed. The experiment was concluded to be positive. The substance was considered to be genotoxic in this assay.

In vivo studies

An in vivo micronucleus assay on mouse bone marrow erythrocytes was conducted under GLP to OECD TG 474. A maximum tolerable dose of 500 mg/kg bw was established in preliminary tests with two female and two male NMRI mice. The main study was performed with male mice, and six animals per group received a single oral dose by gavage of 125, 250 or 500 mg/kg bw in corn oil. Ten animals in the solvent control group received a single oral dose of corn oil, and five animals were administered the positive control substance cyclophosphamide at 40 mg/kg bw in distilled water. Clinical signs of toxicity were observed in the group receiving 500 mg/kg bw, which was indicative of systemic exposure to the test substance. The bone marrow of treated animals was collected 24 hours or 48 hours after oral administration. At least one slide was prepared from each bone marrow sample following standard procedures. All slides were evaluated for the occurrence of micronuclei. Per animal at least 6000 polychromatic erythrocytes (PECs) were scored for micronuclei. The test substance was not cytotoxic to erythrocytes in the bone marrow. There was no biologically relevant or statistically significant increase in the number of detected micronuclei at 24 or 48 hours after a single oral exposure to 125, 250 or 500 mg/kg bw. For all treatment groups the mean values of micronuclei observed after treatment with the substance were below or near the corresponding vehicle control value and well within the the 95% control limit of the historical vehicle control data. The substance was considered to be non-genotoxic (not aneugenic, not clastogenic) in this in vivo test. Treatment with the positive control substance produced a substantial increase of induced micronucleus frequency, which demonstrated the sensitivity and suitability of the test system to detect genotoxic substances.

Justification for classification or non-classification

Based on the negative outcome of the in vitro bacterial reverse mutation assay with and without metabolic activation and the negative result of the in vivo micronucleus assay in bone marrow erythrocytes of mice the substance is considered as non-mutagenic and does not need to be classified for mutagenicity in accordance with Regulation (EC) No 1272/2008.