1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

negative, in vivo mammalian unscheduled DNA synthesis (rat hepatocytes), Ogorek, 1997 negative, in vivo mammalian erythrocyte micronucleus test, OECD 474, Hertner 1995 negative, in vitro bacterial reverse mutation, OECD 471, Hertner 1995 negative, in vitro bacterial reverse mutation, Hertner 1992 negative (in absence of S9 activation), ambiguous in presence of S9 activation, in vitro mammalian gene mutation test, OECD 476, Ogorek 1996 negative, in vitro mammalian chromosome aberration test, OECD 473, Zeugin 1998 negative, in vitro mammalian unscheduled DNA synthesis (rat hepatocytes), OECD 482, Hertner 1995

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study carried out using scientifically valid protocols equivalent to relevant guidelines; adapted for this endpoint according to REACH Annex XI section 1.1.2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

not applicable

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

not applicable

Principles of method if other than guideline:

According to OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo) indicates DNA repair synthesis after excision and removal of a stretch of DNA containing a region of damage induced by chemical substances or physical agents. The test is usually based on the incorporation of 3H-TdR into the DNA of liver cells which have a low frequency of cells in the S-phase of the cell cycle.
Test substances are generally administered as a single treatment administered orally by gavage. The number of animals should be at least 3 analysable animals per group. Where a significant historical database has been accumulated, only 1 or 2 animals are required for the concurrent negative and positive control groups. At least two dose levels are used, the highest dose as defined as the dose producing signs of toxicity. The highest dose may also be defined as a dose that produces some indication of toxicity in the liver (e.g. pyknotic nuclei).
A limit test may be performed if no adverse effects are observed < 2000 mg/kg unless human data/exposure indicate a need for a higher limit dose level.
Liver cells are prepared from treated animals normally 12-16 hours after dosing. An additional earlier sampling time (normally 2-4 hours post-treatment) is generally necessary unless there is a clear positive response at 12-16 hours. However, alternative sampling times may be used when justified on the basis of toxicokinetic data.
- Freshly isolated mammalian liver cells are incubated usually with medium containing 3HTdR for an appropriate length of time, e.g. 3 - 8 hours are then developed and slides developed and analysed for morphology and signs of overt cytotoxicity.
- Slides should be coded before grain counting. Normally 100 cells are scored from each animal from at least two slides; the scoring of less than 100 cells/animal should be justified. Grain counts are not scored for S-phase nuclei, but the proportion of S-phase cells may be recorded.
- The amount of 3H-TdR incorporation in the nuclei and the cytoplasm of morphologically normal cells, as evidenced by the deposition of silver grains, should be determined by suitable methods.
- Grain counts are determined over the nuclei (nuclear grains, NG) and nucleus-equivalent areas over the cytoplasm (cytoplasmic grains, CG). CG counts are measured by either taking the most heavily labelled area of cytoplasm, or by taking an average of two to three random cytoplasmic grain counts adjacent to the nucleus. Other counting methods (e.g. whole cell counting) may be used if justified.
Examples of positive and negative response criteria are given in OECD Guideline 486. A positive result from the UDS test with mammalian liver cells in vivo indicates that a substance induces DNA damage in mammalian liver cells in vivo that can be repaired by unscheduled DNA synthesis in vitro. A negative result indicates that, under the test conditions, the test substance does not induce DNA damage that is detectable by this test.
This is an accurate outline of the procedures used in this, study no. 961122 Ogorek.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

other: Tif: RAJ (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 150 - 350 g
- Assigned to test groups randomly: Yes.
- Fasting period before study: 12 hrs
- Housing: Individually in makrolon cages before and after treatment
- Diet (e.g. ad libitum): Pelleted, certified standard diet administered ad libitum up to 12 hours before dosing.
- Water (e.g. ad libitum): Tap water ad libitum, drinking water quality of recorded specifications
- Acclimation period: Four days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-75%
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 30/09/1996 To: 14/10/1996

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Mixture of Carboxymethylcellulose (0.5% [w/v] in bidistilled water) and Tween 80 (0.1% [v/v] in bidistilled water); 20 ml/kg
- Justification for choice of solvent/vehicle: A solubility test was not performed. From an earlier in vivo study with this test compound,
a mixture of Carboxymethylcellulose (0.5% [w/v] in H20) and Tween 80 (0.1% [v/v] in H20), was known to be a suitable vehicle for preparation of applicable suspensions of the test substance up to a top dose level of 5000 mg/kg body weight.
- Concentration of test material in vehicle: 62.5 and 250 μg/ml respectively.
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A solubility test was not performed. A mixture of Carboxymethylcellulose (0.5% [w/v] in H20) and Tween 80 (0.1% [v/v] in H20), was known to be a suitable vehicle for preparation of applicable suspensions of the test substance up to a top dose level of 5000 mg/kg body weight from a previous reported study. Further information on the preparation of the dose solutions was not reported other than information on dose levels from a preliminary test.

Animals were treated with the selected doses of the test substance, with the vehicle alone or with the positive control by oral application. The treatment regimen was designed in accordance with published standard protocols. For determining UDS treatment, a period of 16 hours was selected for the groups treated with the test substance and with the vehicle alone. With the positive control substance a time point resulting in an optimal UDS response is usually selected. For DMN this time is known to be about 2 hours.

Duration of treatment / exposure:

16 hours main study group and negative control; 2 hours for positive control.

Frequency of treatment:

Single treatment oral dose

Post exposure period:

Not applicable. Primary hepatocytes were freshly isolated by in situ-collagenase perfusion.

Remarks:

Doses / Concentrations:
1250 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

4 male per group

Control animals:

yes, concurrent vehicle

other: positive control received the promutagen dimethylnitrosamine (DMN, 10 mg/kg, 10 ml/kg) dissolved in water.

Positive control(s):

dimethylnitrosamine
- Justification for choice of positive control(s): Not reported.
- Route of administration: Oral gavage
- Doses / concentrations: 10mg/kg dissolved in water (10 ml/kg)

Tissues and cell types examined:

Primary hepatocytes freshly isolated from rats previously treated with the test substance

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Prior to the study test the maximum tolerated dose (MTD) of the test substance to be applied was determined in a tolerability test. The MTD is defined as the highest dose which does not induce severe toxicity or lethality. In two consecutive steps each one male rat was dosed with 5000 mg/kg. Body weight and signs of toxicity were recorded hourly for the first few hours after application and once the following two days.
The rat survived and showed piloerection on the day of treatment. In the second step of the tolerability test (confirmatory experiment) one male rat was dosed with 5000 mg/kg. Again, the animal survived and piloerection was noted after application. Based on these results the doses of 1250, 2500 and 5000 mg/kg were chosen for the DNA-rapair assay.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
For determining UDS treatment, a period of 16 hours was selected for the groups treated with the test substance and with the vehicle alone. With the positive control substance a time point resulting in an optimal UDS response is usually selected. For DMN this time is known to be about 2 hours.
Primary hepatocytes were freshly isolated by in situ-collagenase perfusion.

DETAILS OF SLIDE PREPARATION:
The procedure was as follows: the liver was perfused in situ through the portal vein. The liver was then carefully excised and placed into a dish containing the formulated wash solution. After opening the Glisson's capsule, the cells were dispersed by gently shaking of the liver in the solution. The cells were then filtered (mesh width of 61 μm) and washed once with the same wash solution (sedimentation rate of 50 g for 3 minutes at 2°C). Finally, the cells were suspended in Williams' medium E and analysed for viability by Trypan blue exclusion. Freshly isolated male rat hepatocytes were cultured. Cells were allowed to attach to the cover-slips during an attachment period of 1.5-2 hours. Unattached cells were then removed by washing with BSS and the cultures were refed with culture medium.
The UDS assay was initiated by adding 10 μCi/ml 3H-thymidine (6-3H-thymidine, specific activity 27-28 Curies/mMol). After 4 hours' incubation the cells were washed twice with BSS and further incubated overnight in renewed medium containing
0.25 mMol/1 cold thymidine. In the morning, the cells were washed twice with BSS and fixed with ethanol/acetic acid, 3/1, v/v. The cover-slips were mounted on microscope slides and prepared for autoradiography. Four slides from each animal were autoradiographed; the remainder were kept in reserve. Preparation and development of autoradiographs was performed in a dark room at 20 °C. Autoradiographic emulsion (diluted with two vol. H2O) was used as film material. Preparing the emulsion and coating the slides was performed in red light conditions. The air-dried slides were exposed in light- and airproof boxes containing desiccants at 4 °C for five days. Autoradiographs were developed, rinsed in acetic acid (1%) and fixed in Hypam solution (diluted 1: 10 in water). The autoradiographs were stained in hematoxylin solution, rinsed in tap water and counter-stained in eosin.

METHOD OF ANALYSIS:
Prior to scoring, the slides were coded. Silver grains over the nuclei and cytoplasm of the hepatocytes were counted using an electronic counter (DOMINO) attached to a microscope (ZEISS) at a magnification of 2000x. Two slides (50 cells/slide) from each animal of the treatment groups and of the respective positive and the negative controls were scored. The number of silver grains over the nuclei (nuclear grain counts) were counted and the mean values with the standard deviations were calculated. The results of the nuclear grain counts were tabulated.
The incorporation of radioactive material in the cytoplasm was determined by counting the silver grains in three cytoplasmic regions adjacent to the nucleus, each with an area equivalent to that of the nucleus (cytoplasmic grain counts). The results were tabulated.
The net nuclear grain counts were calculated by subtracting the average grain count over the cytoplasm from the total over the nuclei. The results were tabulated.
To make sure that not only effects on the majority of the cells within the treated hepatocyte population were recognised but also effects on particularly sensitive subpopulations of the hepatocyte culture, the percentage of cells in repair (percentage of cells with a net nuclear grain count value of 2.0 or more) was calculated for each slide and the results were tabulated.

Cells which were in the DNA-synthesis phase show more than 120 silver grains/nucleus. These cells were excluded from the determination of the silver grain/nucleus count.

OTHER:
To confirm that the animals were actually exposed to the intended test concentrations and to confirm the stability of the test substance in the vehicle used, determination of the concentration of the substance in suspension was performed by the analytical unit. This determination was performed with a low dose and a high dose sample of the first and last segment of the DNA-repair test.

Evaluation criteria:

Criteria for a positive response
The test substance is generally considered to be active in the DNA repair test if one of the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains in relation to their respective vehicle control value show an increase at any dose level and the mean net nuclear value is 2.0 or higher.
2. The percentage of cells in repair show an obvious shift to higher values at any dose as compared to their respective vehicle control value.

Criteria for a negative response
The test substance is generally considered to be inactive in the DNA repair test if the following conditions are met:
1. The mean nuclear counts and the mean net nuclear counts of silver grains as well as the percentage of cells in repair, do not significantly differ from the respective vehicle control value at any dose level.

Exceptions
At the limits of the criteria for a positive or for a negative response, or if the criteria for a positive response are only partially fulfilled, the Study Director decides by experience about the interpretation of the results.

Statistics:

Mean values and the standard deviations calculated. Comparison with historical laboratory mean control data from in vivo/in vitro DNA repair data

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no mortalities; piloerection 2-5 hrs after treatment in limit dose group only

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1250, 2500 and 5000 mg/kg
- Solubility: Not applicable.
- Clinical signs of toxicity in test animals: piloerection 2-5 hrs after treatment in limit dose group (5000 mg/kg) only

Table 1. Group mean net grain count values

Dose / mg/kg	Net nuclear grain count (NG)		Net grain count of cells in repair		Percent of cells in repair (NG >= 2)
	Mean	SD	Mean	SD	Mean	SD
0 Negative Control (Vehicle only)	0	0.2	2.6	0.2	8.8	3.6
10 DMN Positive Control	12.5	1.6	12.5	1.6	100.0	0.0
5000	0.1	0.2	3.0	0.3	11.8	3.4
2500	0.0	0.2	2.8	0.1	11.0	4.1
1250	0.1	0.2	2.6	0.2	10.5	1.7

Hepatocyte preparations of good quality were obtained with all animals from all dosage groups. Viability after perfusion was determined to be between 83 and 95%.

 

Evaluation of the mean nuclear counts of silver grains in the vehicle controls and after treatment with test substance revealed no marked differences (Table 1). In consideration of the silver grain number over the cytoplasm, the mean net nuclear grain counts were calculated and there was no marked deviation between the treatment groups and the vehicle controls. Also the percentage of cells in repair (net nuclear grain count ~ 2.0) revealed no marked differences in comparison with the vehicle controls. By contrast, the positive control DMN (15 mg/kg) yielded a marked increase in the mean nuclear grain counts and the mean net nuclear grain counts in comparison with the negative. The percentage of cells in repair was also clearly increased.

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

Executive summary:

The test substance was investigated for DNA-damaging effects on rat hepatocytes after application in vivo and further investigation of unscheduled DNA synthesis (UDS) in vitro. The assay is designed to measure unscheduled DNA synthesis (UDS, DNA-repair synthesis) in freshly isolated rat liver cells (hepatocytes) as a consequence of DNA-damage induced after in vivo treatment of the animals with the test substance. The advantage of this test system is, that not only metabolites formed in the liver itself, but also those formed in the various other organ and cell systems may have an effect in the hepatocytes. Hepatocytes are isolated by perfusion technique, and unscheduled DNA-synthesis caused by the test substance or its metabolites is detected by reference to the incorporation of 3H-TdR into DNA during in vitro culture. DNA-repair is determined by scoring autoradiographs (counts of silver grains over the nuclei; nuclei with< 120 grains are analysed and counts of silver grains over nuclear-sized cytoplasmic areas). There is ample evidence that occurrence of unscheduled DNA-synthesis is closely correlated with mutagenic or carcinogenic properties of a chemical substance, or both. The substance was suspended in a mixture of carboxymethylcellulose (0.5% [w/v] in bidistilled water) and Tween 80 (0.1% [v/v] in bidistilled water and administered orally at single doses of 1250, 2500 and 5000 mg/kg. Hepatocytes from animals of the treatmentment groups and negative control groups were isolated 16 hours after administration of the test substance. Hepatocytes from animals of the positive control group (dimethylnitrosamine, 15 mg/kg) were isolated 2 hours after application.

In the UDS assay, comparison of the mean nuclear and the mean net nuclear counts of silver grains in the vehicle controls and in the hepatocytes of rats treated with the various doses of the substance revealed no marked deviations. Also the percentage of cells in repair after treatment with the test substance was not significantly shifted when compared with the animals of the vehicle control.

It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro data:

OECD 471, Hertner 1995 - Bacterial reverse mutation test (Ames test):

The study was performed to the requirements of OECD 471, EU Method B.14, MHW (Japan) and U SEPA OTS 798.5265 under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA102, TA1535, TA1537 and E. coli strains WP2 uvrA, in both the presence and absence of S-9 mix.

In the main study the test substance was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9-mix, were included. A separate confirmatory study was performed at a concentration up to 5000 µg/plate. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 and E. coli strains WP2 uvrA in both the presence and absence of S-9 mix.

 

Hertner 1992 – Bacterial reverse mutation test (Ames test) equivalent to OECD 471:

The study was performed under GLP conditions, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli strains WP2 uvrA, in both the presence and absence of S-9 mix.

In the main study the test substance was evaluated at a concentration of up to 5000 µg/plate. Positive controls appropriate for each strain, in the presence and absence of S9-mix, were included. A separate confirmatory study was performed at a concentration up to 2500 µg/plate. The test substance did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of revertant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strains WP2 uvrA in both the presence and absence of S-9 mix. In the confirmatory test the test material did not lead to an increase in the incidence revertants in comparison with the negative control.

 

OECD 476, Ogorek, 1996 – Gene mutation test with Chinese hamster V79 cells:

The study was performed to the requirements of OECD 476 (1997), EC Method B.17 (2000), and US EPA OTS 798.5300 under GLP conditions was performed to evaluate the potential of the test substance to cause gene mutation or clastogenic effects in mammalian cells within in vitro cell mutation assay in Chinese Hamster V79 cells. The test substance was evaluated over a range of concentrations, both in the presence and absence of S9-mix, in two independent mutation experiments.

In the presence of S9 activation, the main experiment showed that comparison of the number of mutant colonies in the controls and in the cultures treated with the test material revealed a slight but statistically significant increase in the mutant frequency at the highest concentration of 100 µg/mL. In the confirmatory experiment, at the same concentration a weak increase in the number of mutant colonies was found, however, this did not fulfil the criteria for a positive response. Due to the lack of a reproducible effect the overall result is negative. In the absence of S9 activation, in both the main and confirmatory experiments, the number of mutant colonies in the controls and in the treated cultures revealed no relevant increase of the mutant frequencies. The positive controls induced appropriate increases in mutant frequency in all mutation experiments, demonstrating the activity of the S9-mix and that the assay had performed satisfactorily. It was concluded that, under the conditions of this assay, the test substance was not mutagenic in Chinese Hamster V79 cells treated in vitro in the absence of S9 activation; however the results are ambiguous in the presence of S9 activation.

 

OECD 473, Zeugin 1998 – Mammalian chromosome aberration test with Chinese hamster ovary cell line:

The study was performed to the requirements of OECD 473, EC Method B.10, MAFF Japan and US EPA OTS 798.575 under GLP conditions. A chromosome aberration test with Chinese Hamster was performed to evaluate the potential of the test substance to cause gene clastogenic effects in mammalian cells. The test substance was evaluated over a range of concentrations, both in the presence and absence of S9-mix. An initial toxicity test was performed to determine the concentrations to be used for the main tests. In total for the original and confirmatory tests, three experiments without metabolic activation (twice 18 hours treatment and once 42 hours treatment each) and three trials with metabolic activation (twice 3 hours treatment/15 hours recovery and once 3 hours treatment/39 hours recovery) were performed. A supplementary experiment was also performed with metabolic activation (once 3 hours treatment/15 hours recovery and once 3 hours treatment/39 hours recovery). In the original and confirmatory tests without S9 activation, no cytotoxicity was observed no increases in the number of metaphases with chromosomal aberrations were observed. In the original experiment performed with metabolic activation (3 hours treatment/15 hours recovery), final concentrations higher than the respective highest concentration 62.5 and 125.0 μg/ml of culture medium could not be scored due to toxicity; cytotoxicity was observed at these concentrations. In the confirmatory experiment with metabolic activation (3 hours treatment/39 hours recovery ), a final concentration higher than 250 μg/ml of culture medium could not be achieved due to solubility limitations. A supplementary experiment (3 hours treatment/ 15 and 39 hours recovery) was performed to confirm the results obtained in the confirmatory experiment at the long recovery time. With metabolic activation no increase in the number of metaphases with chromosomal aberrations were seen at 3 hours treatment and 15 hours recovery. The slight increase in one isolated experiment at the long recovery time (3 hours treatment and 39 hours recovery) could not be reproduced in a supplementary experiment and was therefore not considered to be indicative of a clastogenic activity of the test compound. These isolated effects appeared only at concentrations which proved considerable toxic in experiments with shorter recovery time (15 hours) and were therefore attributed to toxicity. The positive controls induced the appropriate increases of clastogenic effects. Under the conditions of this experiment, the test material did not cause clastogenic effects in Chinese hamster ovary cells in vitro.

 

OECD 482, Hertner 1995 – Mammalian unscheduled DNA synthesis: rat hepatocytes:

The study was performed to the requirements of OECD Guideline 482 and US EPA OTS 798.5550 under GLP conditions. The study completed an investigation on the test substance or its metabolites to induce DNA damage in primary hepatocytes in vitro, freshly isolated from male rats. This test system permits the detection of unscheduled DNA synthesis as a consequence of DNA damage induced by chemical substances or by their metabolites.

The substance was dissolved in DMSO and tested at concentrations from 0.98 to 31.25 μg/ml (original experiment) and from 1.56 to 50.0 μmol/l (confirmatory experiment). Concentrations of 62.5 μg/ml and higher proved to be toxic (no viable cells on the slides). 2-acetylaminofluorene (45 μmol/l) was used as a positive control. In both experiments performed, the test substance did not induce a significant increase in the DNA repair activity of primary rat hepatocytes. No evidence of induction of DNA damage the substance or by its metabolites was obtained that could be interpreted as suggestive of a genotoxic property of the test substance in vitro.

 

In vivo data:

OECD 474, Hertner 1995 - Mammalian erythrocyte micronucleus assay: mouse:

The study was performed to the requirements of OECD 474, EU Method B.12, MITI (Japan) and US EPA OTS 798.5395 under GLP conditions and to evaluate the potential mutagenicity of the test substance in a mammalian erythrocyte micronucleus assay. A tolerability test was performed to determine the test concentrations for the definitive micronucleus study. In the definitive test, 10 test animals received the test substance at a concentrations of 1250, 1000, 625 and 312.5 mg/kg. Test animals also received bidistilled water (vehicle controls) and cyclophosphamide (positive control). After treatment for 16, 24 and 48 hours, test animals were sacrificed and the bone marrow was harvested and stained for analysis. Females for the 24 and 48 hours sampling time were treated with the high dose of 1250 mg/kg. This dose proved heavily toxic and 7 out of 13 treated animals were found dead after 4 to 5 hours. The remaining females showed no symptoms of toxicity. Due to this serious toxicity the dose level for the 16 hours sampling group (females) and the high dose for the male animals (all sampling times) was reduced to 1000 mg/kg. At all sampling times (16, 24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the· respective doses of test material as compared with the negative control animals. It was concluded that, under the conditions of this assay, the test substance did not cause clastogenic or aneugenic effects.

 

Ogorek, 1995 - Mammalian unscheduled DNA synthesis: rat hepatocytes:

The test substance was investigated for DNA-damaging effects on rat hepatocytes after application in vivo and further investigation of unscheduled DNA synthesis (UDS) in vitro. The assay is designed to measure unscheduled DNA synthesis (UDS, DNA-repair synthesis) in freshly isolated rat liver cells (hepatocytes) as a consequence of DNA-damage induced after in vivo treatment of the animals with the test substance. The advantage of this test system is, that not only metabolites formed in the liver itself, but also those formed in the various other organ and cell systems may have an effect in the hepatocytes.

The substance was suspended in a mixture of carboxymethylcellulose (0.5% [w/v] in bidistilled water) and Tween 80 (0.1% [v/v] in bidistilled water and administered orally at single doses of 1250, 2500 and 5000 mg/kg. Hepatocytes from animals of the treatment groups and negative control groups were isolated 16 hours after administration of the test substance. Hepatocytes from animals of the positive control group (dimethylnitrosamine, 15 mg/kg) were isolated 2 hours after application.

In the UDS assay, comparison of the mean nuclear and the mean net nuclear counts of silver grains in the vehicle controls and in the hepatocytes of rats treated with the various doses of the substance revealed no marked deviations. Also the percentage of cells in repair after treatment with the test substance was not significantly shifted when compared with the animals of the vehicle control. It is concluded that, under the given experimental conditions, no evidence of induction of DNA damage by the substance or by its metabolites was obtained that could be interpreted as suggestive of genotoxic properties of the substance.

Justification for selection of genetic toxicity endpoint
Study selected is an in vivo study (Klimisch 1) representative of the substance itself and metabolites (indirect action) and is clearly consistent with the weight of evidence for all in vivo and in vitro studies.

Justification for classification or non-classification

The substance does not meet classification criteria under EU Directive 67/548/EEC for mutagenicity

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity