[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an OECD 471 study the test substance showed no evidence of mutagenic activity.

 

In an OECD 473 study the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations or of causing an increase in numerical aberrations in the form of polyploidy (or endoreduplication), in this in vitro cytogenetic test system, under the experimental conditions described.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 August 2015 to 15 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: See below.

Version / remarks:

Official notice of MHLW, METI and MOE (31 March 2011)
YAKUSHOKUHATSU 0331 No 7
SEIKYOKU No 5
KANPOKIHATSU No 110331009.
Official Notice of J MOL (16 September 1999).

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and
5,6-benzoflavone to stimulate mixed-function oxidases in the liver

Test concentrations with justification for top dose:

Concentrations with high ionic strength and osmolality may cause chromosomal aberrations
(Galloway et al. 1987). Therefore, the osmolality of the test substance in medium was tested
at concentrations of 1800, 3000 and 5000 micro g/mL. Concentrations of 3000 and 5000 micro g/mL caused a change of greater than 50 mOsm/kg when compared with the vehicle control.
No fluctuations in pH of the medium were observed at 1800, 3000 and 5000 micro g/mL of more
than 1.0 unit compared with the vehicle control. As changes of greater than 50 mOsm/kg
were observed at higher concentrations, the maximum final concentration tested in the
preliminary toxicity test was 1800 micro g/mL.

In the main test, justification for the highest analysed concentration was determined by
cytotoxicity for all treatment groups.

Vehicle / solvent:

Prior to commencing testing, the solubility of the test substance in vehicle compatible with this test system was assessed. It was found to be soluble at 500 mg/mL in ethanol.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Preliminary test:
A culture of CHL cells in a 75 cm2 flask was harvested as follows: the supernatant medium was removed and 8 mL Accutase™ (cell detachment solution) was added. The cells were then examined microscopically, and if “rounded”, they were dislodged and a sample taken for counting.
The cells were then re-suspended in an appropriate volume of culture medium to give 2 x 104
cells/mL. Aliquots (5 mL) of the cell suspension were added to 25 cm2 tissue culture flasks and were incubated at 34-39 deg.C in an atmosphere containing 5% carbon dioxide.

Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment
with the test substance for each test condition. No positive control cultures were prepared.

After approximately 24 hours, the culture medium was removed from each culture and replaced with fresh medium, allowing for the volume of S9 mix where appropriate.

The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control.

At the end of the 3-hour treatment period, cultures were examined for the presence of precipitate. Cultures were washed in saline and fresh medium was added to the flasks which were then incubated (for approximately 12 hours) until the scheduled harvest time.

At the end of the 15-hour treatment period, cultures were examined for the presence of precipitate.

Main test:
The procedure for the main test was the same as that for the preliminary test, with the following exceptions; positive control cultures were included for all tests, duplicate cultures were prepared for all cultures and two slides were prepared for selected cultures.

3-hour treatment in the absence of S9 mix - The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C, at final concentrations of 0.1 and 0.2 µg/mL, was added to duplicate cultures.

Following 3-hour treatment, medium was removed from the flasks and discarded. Cells were
washed in saline before fresh medium was added to the cells. They were then incubated for a
further 12 hours. The cultures were then harvested and slides prepared.

3-hour treatment in the presence of S9 mix - For treatments in the presence of S9 mix, 1 mL of S9 mix was added to give a concentration of 1% v/v in the final test medium.

RThe test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Cyclophosphamide at final concentrations of 5 and 10 µg/mL was added to duplicate cultures.

Following 3-hour treatment, medium was removed from the flasks and discarded. Cells were
washed in saline before fresh medium was added to the cells. They were then incubated for a
further 12 hours. The cultures were then harvested and slides prepared.

15-hour treatment in the absence of S9 mix - The test substance was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C, at final concentrations of 0.1 and 0.2 µg/mL, was added to duplicate cultures.

Following the end of the treatment period the cultures were harvested and slides prepared.

Evaluation criteria:

Providing that all of the acceptance criteria have been met, the test substance was considered
to be clearly positive if, in any of the experimental conditions examined:

At least one of the test concentrations exhibited a statistically significant
increase compared with the concurrent vehicle control.

The increase was dose-related when evaluated with an appropriate trend
test.

Any of the results were outside the distribution of the historical negative
control data.

If all of these criteria were met, the test substance was considered able to induce chromosome
breaks and/or gain or loss in the test system.

Providing that all of the acceptance criteria have been met, a negative response was claimed
if, in all of the experimental conditions examined:

None of the test concentrations exhibited a statistically significant increase
compared with the concurrent negative control.

There was no concentration-related increase when evaluated with an
appropriate trend test.

All results were inside the distribution of the historical negative control
data.

If all of these criteria are met, the test substance was considered unable to induce
chromosome breaks and/or gain or loss in the test system.

The Study Director used scientific judgement to classify data that did not fall into either of
the above categories.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence and presence of S9 mix following 3 and 15 hours in the absence of S9 mix and
for 3 hours in the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated
metaphase cells were observed during metaphase analysis, under any treatment condition,
when compared to the vehicle control.

Both positive control compounds caused statistically significant increases in the proportion of
aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Conclusions:

In an OECD 473 study the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations or of causing an increase in numerical aberrations in the form of polyploidy (or endoreduplication), in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of the test substance to induce chromosomal aberrations in Chinese Hamster Lung (CHL) cells cultured in vitro.

CHL cells were grown and sub-cultured in tissue culture medium at 34-39 deg.C in an atmosphere containing 5% carbon dioxide. They were then incubated with the test substance in both the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included (where applicable). Two hours before the end of the incubation period, cell division was arrested using Colcemid® , the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

The study consisted of a preliminary toxicity test and a main test. In both types of tests the cells were treated for 3 and 15 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. The Relative Increase in Cell Counts (RICC) was assessed for all cultures to determine cytotoxicity.

In the main test, justification for the highest analysed concentration was determined by cytotoxicity for all treatment groups.

The following test substance concentrations were selected for metaphase analysis:

In the absence of S9 mix, 3-hour treatment: 25, 87.5 and 100 µg/mL
In the presence of S9 mix, 3-hour treatment: 50, 200 and 225 µg/mL
In the absence of S9 mix, 15-hour treatment: 6.25, 12.5 and 25 µg/mL

In the absence and presence of S9 mix following 3 and 15 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix, the test substance caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the
vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

 

In an OECD 473 study the test substance has shown no evidence of causing an increase in the frequency of structural chromosome aberrations or of causing an increase in numerical aberrations in the form of polyploidy (or endoreduplication), in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 June 2015 to 2 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

S9 fraction was prepared from male Sprague-Dawley derived rats, dosed with phenobarbital/5,6-benzoflavone to stimulate mixed-function oxidases in the liver.

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Vehicle / solvent:

Ethanol

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

First test:
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate),
positive control or vehicle control were placed in glass tubes. The vehicle control was ethanol. S9 mix (0.5 mL) or 0.1 M pH 7.4 sodium phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar.

Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37 deg. C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

Any toxic effects of the test substance may be detected by a substantial reduction in mean revertant colony counts, by a sparse or absent background bacterial lawn, or both. In the absence of any toxic effects, the maximum concentration selected for use in the second test is the same as that used in the first. If toxic effects are observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition are present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate is observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be included in the second test.

Second test:
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for ethanol, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 µg/plate.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.

If exposure to a test substance does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by (Mahon et al 1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Occasionally, these criteria may not be appropriate to the test data and, in such cases, the
Study Director would use his/her scientific judgement.

Statistics:

No statistical analysis is performed.

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

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Precipitate was observed on all plates containing the test substance at 5000 µg/plate.

Conclusions:

In an OECD 471 study the test substance showed no evidence of mutagenic activity.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), were exposed to the test substance diluted in ethanol. Ethanol was also used as a vehicle control.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. Both tests were standard plate incorporation assays.

Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Toxicity (observed as thinning of the background lawn and/or a reduction in revertant colony numbers) was seen in strains TA100, TA1535 and TA1537 and WP2uvrA (pKM101) following exposure to the test substance at various concentrations from 500, 1500 and 5000 µg/plate in the first test.

Toxicity (observed as thinning of the background lawn and/or a reduction in revertant colony numbers) was seen in strains TA100, TA1535 and TA1537 in the absence of S9 mix at various concentrations from 500, 1500 and 5000 µg/plate and in strains TA100, TA1537 and WP2 uvrA (pKM101) in the presence of S9 mix following exposure to the test substance at concentrations of 1500 and/or 5000 µg/plate in the second test.

Precipitate was observed on all plates containing the test substance at 5000 µg/plate in both tests.

No evidence of mutagenic activity was seen at any concentration of the test substance in either mutation test.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

It was concluded that the test substance showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

During an OECD 474 study and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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:

9 October 2006 to 30 January 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

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. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of
Acclimatisation: 8 - 10 weeks
Acclimatisation: minimum 5 days

Initial body weight at Start of Treatment - males mean value 35.6 g (SD ± 1.8 g), females mean value 29.8 g (SD ± 1.6 g).

According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

The animals were distributed into the test groups at random and identified by cage
number.

Environmental Conditions
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.

Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment:
- temperature 22 ± 3 °C
- relative humidity 30 - 75 %
- artificial light 6.00 a.m. - 6.00 p.m.

Route of administration:

intraperitoneal

Vehicle:

30% DMSO / 70% corn oil

Duration of treatment / exposure:

The volume administered intraperitoneally (i.p.) was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Frequency of treatment:

Single administration

Post exposure period:

24 h and 48 h

Dose / conc.:

250 mg/kg bw (total dose)

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Ten animals (5 males, 5 females) per test group were evaluated as described.

The remaining 6th animal of each sex in the respective test group test group is usually
evaluated in case an animal dies in its test group spontaneously.

Control animals:

yes, concurrent vehicle

Positive control(s):

CPA; Cyclophosphamide

Solution prepared on day of administration.

Tissues and cell types examined:

Bone marrow cells were collected for micronuclei analysis.

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 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:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously.

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.

A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of polychromatic erythrocytes was slightly decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control indicating that the test substance had cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with test substance were near to the value of the vehicle control group and within the historical control range.

40 mg/kg b.w. cyclophosphamide administered i.p. was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusions:

During an OECD 474 study and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in 30% DMSO / 70% corn oil, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 250, 500 and 1000 mg/kg b.w..
48 h preparation interval: 1000 mg/kg b.w..

The highest dose (1000 mg/kg b.w.) was estimated by a pre-experiment to be suitable as the high dose. In the main study 1 male died after treatment with this dose.

After treatment with the test item the number of PCEs was slightly decreased as
compared to the mean value of PCEs of the vehicle control thus indicating that the test substance exerted 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 item and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered i.p. was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, the test substance is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)