Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102, E.coli WP2 uvrA: negative with and without metabolic activation (according to OECD 471) (reference 7.6.1-1)

Chromosome Aberration Assay: Treatment of the V79 cell line cultures with the test item at cytotoxic and non-toxic concentrations significantly increased the proportion of cells with aberrant chromosomes in the presence of S9 mix (reference 7.6.1 -2)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-01-25 to 2000-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
29 Decembre 1992
Deviations:
no
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 TA 102
Remarks:
Escherichia coli WP2 uvrA pkM101
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
- source of S9
Male (Wistar) rats (aged 6-8 weeks) were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil (MERCK, Article-No. 6175). Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice-cooled, sterilized beakers containing 0.15 M KCl. The animals were prepared unfasted.

- method of preparation of S9 mix
The livers were homogenized in a sterile glass potter homogenizer'-containing 3 mL of 0.15 M KCl per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen in liquid nitrogen and stored at -196 °C.

- concentration or volume of S9 mix in the final culture medium : 0.5 mL/plate (S9 homogenate 0.1 mL (1st series); 0.3 mL (2nd series))

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch.
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)
2nd series: 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
DAUN
Positive control substance:
other: Daunomycin
Remarks:
TA 98 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
ENNG
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
TA 100, 1535, WP2 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CUM
Positive control substance:
cumene hydroperoxide
Remarks:
TA 102 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
9-AA
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 (without S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2-AA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
TA 98, TA 100, TA 1535, TA 1537, WP 2 (with S9 mix)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
B(a)P
Positive control substance:
benzo(a)pyrene
Remarks:
TA 102 (with S9 mix)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
None observed.

STUDY RESULTS
Ames test:
- Signs of toxicity : No cytotoxicity was observed.
- Mean number of revertant colonies per plate and standard deviation : Refer to attached background material
Conclusions:
With and without addition of S9 mix as external metabolising system, the test substance was not mutagenic under the experimental conditions described.
Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM 101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test substance was dissolved in dimethyl sulfoxide and tested at the following concentrations:

 

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

2nd series: 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

 

Precipitation of the test material on the agar plates and toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system, the test substance was not mutagenic under the experimental conditions described.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-02-07 to 2000-06-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
21st July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
29 December 1992
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: from lung tissue of a male Chinese hamster

For cell lines:
- Absence of Mycoplasma contamination: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Dulbecco's Minimal Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375 %), antibiotics (neomycine 0.015 %), and 10 % fetal calf serum (FCS). All incubations were performed at +37° C in a 4-5 % carbon dioxide atmosphere (100 % humidity).
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
- source of S9
Five days before sacrifice, male Wistar rats (Thomae, Biberach, Germany), weighing 100-160 g, aged 7-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (MERCK KGaA, Article No. 6175). The animals received drinking water and a standard diet ad libitum. On day 5, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing PBS-HEPES buffer (phosphate and HEPES (20 mM) buffered saline, pH 7.4).

- method of preparation of S9 mix
After homogenization in a sterile glass potter homoge-nizer with a Teflon pestle the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 15 minutes at about +4°C Finally, the supernatant fluid (termed S9) was frozen and stored in liquid nitrogen.

- concentration or volume of S9 mix in the final culture medium: 0.1 mL

- quality controls of S9
Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The bacterial mutagenicity (according to Ames et al., 1975) of 2-aminoanthracene, benzo(a)pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for various bacterial strains with all positive controls are used as an acceptance criterium for each S9-batch.
Test concentrations with justification for top dose:
1st series: 281, 500 and 889 µg/mL (with S9 mix)
2nd series: 158, 281, 500, 889 and 1580 µg/mL (with S9 mix)
1st and 2nd series: 88.9, 281, 889 µg/mL (without S9 mix)

Dose justification
The top concentration should inhibit mitotic activity by more than 50% or exhibit some other indication of cytotoxicity. If not toxic, the test material should be tested up to the solubility limit, or up to a maximum concentration of 5 mg/mL. Morphologic changes of the chromosome structure, either fringed or insufficiently spread chromosomes, and a reduction in the mitotic index were used in the present study as parameters indicating the cytotoxicity of the test material.
In the first experimental series, concentrations ranging from 5.00 to 5000 µg test material per mL were tested. The test substance markedly reduced the mitotic index of the V79 cells at the concentration of 889 µg/mL. At higher concentrations no metaphases were present. In the second series, meta-phases were found up to the concentration of 1580 µg/mL.
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CPA
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
EMS
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 5 hours
Fixation time:
With S9-mix: 2.5 h
Without S9-mix: 2.5 and 3.5 h

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : 4 for solvent control; 2 for test substance treatment
- Number of independent experiments : 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours

FOR CHROMOSOME ABERRATION
- Spindle inhibitor: 22 hours after start of the treatment, colchicine is added (to yield a final concentration of 0.1 µg/mL) and the cultures are incubated for another 3 hours
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
The medium was replaced by KCl-solution (0.4 %) for hypotonic treatment. For fixation of cells fixative (methanol: acetic acid, 3:1) was added and then renewed 2 times. The slides are then stained in aceto-orcein, immersed in xylene and made permanent with EntellanR.
- Number of cells spread and analysed per concentration: total of 100 well spread metaphases was examined per culture (slide)
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
Structural chromosome alterations are scored as follows:
Gap:Achromatic region in chromatid(s) not greater than the width of a chromatid. Scored as gap (chromatid) or isogap (chromosomal).
Break:Achromatic region in chromatid(s) greater than the width of a chromatid or a discontinuity with displacement. Scored as break (chromatid) or isobreak (chromosomal).
Exchange: Aberrations arising from an exchange between one or two chromatids. These may be chromosome or chromatid interchanges. In studies of this type, where full karyotyping and chromosome banding are not performed, only asymmetrical or chromatid exchanges will normally be recognized.
Multiple aberrations: Cells with more than five aberrations, gaps excluded.
Specific aberrations: Atypic chromosomes and pulverized metaphases.
The position of each aberrant metaphase is recorded by Vernier reading, and photographs of the first five aberrant metaphases of each slide are taken.
- Determination of polyploidy: The frequency determination of polyploid cells is based on scoring 1000 mitoses per slide, and estimation of the mitotic index on scoring 1000 cells per slide.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)
Evaluation criteria:
A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
- a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
- a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non-toxicological investigations.
Statistics:
For all groups, mean values were calculated for the following parameters:
- the percentage of observed aberrant metaphases/culture (gaps included),
- the percentage of observed aberrant metaphases/culture (gaps excluded),
- the number of mitoses per 1000 cells/culture
- the number of polyploid cells per 1000 cells/culture.
For further statistical analysis the numbers of aberrant metaphases (gaps excluded) were used.
Pairwise comparisons within each experimental series
Each treatment group was compared with the negative control. For the comparisons the Exact Fisher Test (Sachs, 1984) was performed against one-sided alternatives.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
889 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
889 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: A change in the pH of the cell culture medium did not occur in the dose range tested.
- Data on osmolality: A change in the osmotic value of the cell culture medium did not occur in the dose range tested.
- Precipitation and time of the determination: Precipitation of the test material was seen at concentrations > 500 µg/mL when given to the culture medium. This precipitate remained until the end of the exposure time.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
For the positive control (CPA) a clear statistically significant increase of aberrant metaphases was found in both, the first and second experiment.

- Results from cytotoxicity measurements:
In the first experimental series, concentrations ranging from 5.00 to 5000 µg test material per mL were tested. The test item markedly reduced the mitotic index of the V79 cells at the concentration of 889 µg/mL. At higher concentrations no metaphases were present. In the second series, metaphases were found up to the concentration of 1580 µg/mL.
- Genotoxicity results (see attachment)
The mean values of aberrant metaphases (gaps excluded) in the test item-treated groups of both experiments ranged from 8.5 - 16.5 %, as compared to 1.25 and 2.0 % in the negative controls. The biostatistical analysis for the parameter aberrant metaphases (gaps excluded) showed significantly elevated aberration values for the test material. Furthermore, these aberration rates were above the usual range for the V79 cell line used in this laboratory. No treatment-related increase of polyploid cells was observed.

HISTORICAL CONTROL DATA
see attachment
Conclusions:
Treatment of the V79 cell line cultures with the test item at cytotoxic and non-toxic concentrations significantly increased the proportion of cells with aberrant chromosomes in the presence of S9 mix.
Executive summary:

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. The following experimental conditions were selected in the presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Aroclor 1254):

No. of slides per concentration:

Solvent control: 4

Others: 2

No. of metaphases evaluated per slide: 100

Preparation time: 25 hours

Exposure time: 5 hours

Concentrations evaluated:

First series: 281, 500, and 889 µg/mL

Second series: 158, 281, 500, 889 and 1580 µg/mL

Positive control: 4.00 µg Cyclophosphamide (CPA) / mL

The positive control compound, CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations.

In the first experimental series, the test item markedly reduced the mitotic index of the V79 cells at the concentration of 889 µg/mL. At higher concentrations no metaphases were present. In the second series, metaphases were found up to the concentration of 1580 µg/mL.

The test item did not lead to an increase in the number of polyploid cells. However, the substance significantly increased the proportion of cells with aberrant chromosomes as compared to the actual controls. This result was confirmed in the two independently performed experimental series.

In conclusion, the test item was clastogenic in this in vitro test system in the presence of S9 mix..

An additional test was performed without exogenous metabolizing system with the following conditions:

Exposure times: 5 hours (1st series) 25 and 35 hous (2nd series)

Preparation times: 25 and 35 hours

Concentrations evaluated: 88.9, 281 and 889 µg/mL

Positive control: 500 µg Ethylmethansulfonat (EMS) / mL

The positive control compound EMS, induced the expected clear increase in the proportion of cells with chromosomal aberrations.

The test item precipitated in the culture medium at the highest concentration tested, i.e. 889 (µg/mL). A reduction in the mitotic index of the V79 cells was seen at the concentration of 889 µg/mL in the 1stexperimental series.

Treatment of V79 cell cultures with the test item weakly increased the proportion of cells with aberrant chromosomes at two dose levels in one experimental series. These effects, however, were not seen in the first series performed. The test item showed equivocal effects of uncertain biological relevance in this in vitro test system in the absence of S9 mix.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines and the OECD Guideline for Testing of Chemicals No. 474. The substance was not mutagenic in the micronucleus test in male rats under conditions where the positive control exerted potent mutagenic effects (reference 7.6.2 -1).

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:
2002-05-13 to 2002-05-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
8 June 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, 33178 Borchen, Gartenstraße 27, Postfach 1161, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: mean 177 g
- Assigned to test groups randomly: yes, under following basis: according to a random list which had been provided by the Rando 96 program developed and used at Merck KGaA, Darmstadt.
- Housing: individually, Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm)
- Diet: ad libitum, standard diet (Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 49 - 52
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
Miglyoloil 812 (10 mL/kg body weight)
Duration of treatment / exposure:
Dosing of the animals of different groups was staggerred over one to three day intervals to allow for sufficient sample preparation time.
Frequency of treatment:
once
Post exposure period:
none
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
316 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
Male: 100 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 316 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide: 16.5 mg CPA were dissolved in 10 mL Aqua pro injectione directly before use
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose given in the present study was selected to produce signs of toxicity but no mortality. In a preliminary dose-finding experiment 6 male and 6 female rats treated intraperitoneally with 2000 mg/kg bw, showed clear toxic effects and two male and one female rat died. In a second dose-finding experiment, 5 male and 5 female rats treated intraperitoneally with 1000 mg/kg bw showed clear toxic effects (ptosis, dyspnea, diarrhea, prone position and locomotor disturbance) but not mortality. For these reasons, the dose of 1000 mg/kg body weight was selected as the highest doses for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of VlO = 3.16.

TREATMENT AND SAMPLING TIMES
Dosing of the animals of different groups was staggerred over one to three day intervals to allow for sufficient sample preparation time.
For the highest test item dose groups (1000 mg/kg) preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the lower dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment.

DETAILS OF SLIDE PREPARATION:
Immediately after the rats had been killed by CO2, one femur of each animal was dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum (Biochrom, Berlin, Germany) with the aid of a syringe, and suspended in the serum. This suspension was filtered through cellulose according to Romagna (1988) and centrifuged for 5 min at 150 x g. The sediment was then resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension.
After 3 hours of drying, the slides were stained according to a modified Giemsa-staining method described by Gollapudi and Kamra (1979) using Giemsa's solution (Merck, Darmstadt, Germany) with Weise buffer solution (Merck, Darmstadt, Germany) and mounted in Entellan (Merck, Darmstadt, Germany).

METHOD OF ANALYSIS:
A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250x). Round particles with about 1/20 - 1/5 the diameter of an erythrocyte that stained violet, like nucleic material, were scored as micronuclei. They were differentiated from granules by thorough examination at different optical levels. Only erythrocytes with a distinct bluish touch were evaluated as polychromatic. For determination of the quotient of normochromatic to polychromatic erythrocytes, both erythrocyte stages were screened for micronuclei and counted separately up to a total of 1000 erythrocytes per animal. Then counting was limited to polychromatic erythrocytes. Normochromatic erythrocytes with micronuclei, however, observed during scoring were registered also. Thus, based on this value and on the quotient, the number of micronucleated normochromatic erythrocytes per 1000 could be extrapolated. After termination of the microscopic evaluation, the data were loaded into a computer-aided program.
Evaluation criteria:
A positive effect in this test system is defined by the occurrence of mean MN-PCE values of a treatment group which are statistically significantly higher than those of the actual negative control. A prerequisite for this is, however, that these values are above those predetermined as historical negative controls of our laboratory (Historical data).
An indispensable prerequisite for evaluating the results of such investigations is the occurrence of significant positive effects in the actual positive control group.
A test material showing no positive effect in the main study is defined as a non- mutagen in this test system. In this case the study is terminated.
If a positive effect in a single test group occurs (i.e. dose-independently), a repeat experiment has to be considered. In case that no positive effects occur in that experiment the test material is defined as a non-mutagen. The single positive effect of the first experiment is interpreted as a randomly occurring event of no biological significance.
A test material is defined as mutagenic in this system if dose-related and/or single, reproducible (in independent experiments) positive effects occur. Establishment of dose-dependent effects of the test material is preferable. For this reason, if a positive effect occurs in a study in which a single, limit dose of 2000 mg/kg has been appli-cated, 3 different test material doses have to be administered in the supplementary experiment. The above mentioned criteria for a negative or positive test result apply for this experimental design like wise.
If borderline cases occur, the decision on further procedures should be based on a scientific evaluation of all available results including the toxicokinetic data.
Statistics:
Descriptive statistics
For all groups, mean values were calculated of the following parameters:
NCE/PCE - number of normochromatic erythrocytes (NCE) / number of polychromatic erythrocytes (PCE) / animal
MN-NCE - number of micronuclei-containing cells / 1000 NCE / animal
MN-PCE - number of micronuclei-containing cells / 1000 PCE / animal
For the parameter body weight the mean values and the relative body weight gains to the preceding mean values were calculated.

For further statistical analysis the number of micronuclei-containing polychromatic erythrocytes (MN-PCE), normochromatic erythrocytes (MN-NCE) and the quotient of NCE/PCE per animal was used.

Pairwise comparison
Each treatment group was compared to the negative control.
For comparisons of micronuclei-containing polychromatic and normochromatic erythrocytes, the exact Mann-Whitney-test was used against one-sided alternatives. The p-values (exact significance one-sided) of these comparisons are presented.
For comparisons of the quotient of NCE/PCE, the Dunnett's t-test was used against two-sided alternatives. The p-values (significance) of these comparisons are presented for those dose groups that showed a higher mean value than the negative control.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 1000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a preliminary dose-finding experiment 6 male and 6 female rats treated intraperitoneally with 2000 mg/kg bw, showed clear toxic effects and two male and one female rat died. In a second dose-finding experiment, 5 male and 5 female rats treated intraperitoneally with 1000 mg/kg bw showed clear toxic effects (ptosis, dyspnea, diarrhea, prone position and locomotor disturbance) but not mortality.

RESULTS OF DEFINITIVE STUDY
2000 polychromatic erythrocytes per animal, using 5 males per group, were evaluated. The negative control (solvent) values were all in the expected range predetermined as historical controls of the laboratory. For the positive control, a statistically significant increase in polychromatic erythrocytes with micronuclei was established (p < 0.01). No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed. For the number of normochromatic cells with micronuclei, calculated per 1000 normo-chromatic erythrocytes by means of the above mentioned quotient, no increase was observed.
Quotient normochromatic /polychromatic erythrocytes: No relevant treatment-related variation was observed
A relevant treatment-related decrease in body weight, in comparison to the solvent control animals, was not observed.
Conclusions:
No increase in micronucleated polychromatic erythrocytes was observed in this micronucleus test in male rats after a single intraperitoneal administration of the test item. According to the predetermined criteria for interpretation of results the substance is not mutagenic in this test system.
Executive summary:

A micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines and the OECD Guideline for Testing of Chemicals No. 474.

The substance was given once intraperitoneally by gavage to male rats, at doses of 100, 316 and 1000 mg/kg body weight. Rats of the negative control group received the solvent alone, i.e. an intraperitoneal dose of 10 mL Miglyoloil / kg body weight. The animals of the positive control group were treated with an oral dose of 16.5 mg cyclophosphamide/kg body weight.A total of 30 animals was used.

Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the highest dose groups (1000 mg/kg) preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the lower dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. For microscopic investigation one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micro-nucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.

The highest test material dose induced clear clinical signs of toxicity. No relevant treatment-related decrease in body weight (in comparison to the solvent control) occurred and no relevant variation was observed for the quotient normochromatic : polychromatic erythrocytes.

The mean numbers of polychromatic erythrocytes with micronuclei for the negative control (solvent) were all in or very close to the expected range predetermined by historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.

No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

The substance was not mutagenic in the micronucleus test in male rats under conditions where the positive control exerted potent mutagenic effects.

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

Additional information

Bacterial reverse mutation assay

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM 101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. The test substance was dissolved in dimethyl sulfoxide and tested at the following concentrations:

 

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

2nd series: 50.0, 158, 500, 1580 and 5000 µg/plate (± S9 mix)

 

Precipitation of the test material on the agar plates and toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

With and without addition of S9 mix as the external metabolizing system, the test substance was not mutagenic under the experimental conditions described (reference 7.6.1 -1).

Chromosome Aberration Assay

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro. The following experimental conditions were selected in the presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Aroclor 1254):

No. of slides per concentration:

Solvent control: 4

Others: 2

No. of metaphases evaluated per slide: 100

Preparation time: 25 hours

Exposure time: 5 hours

Concentrations evaluated:

First series: 281, 500, and 889 µg/mL

Second series: 158, 281, 500, 889 and 1580 µg/mL

Positive control: 4.00 µg Cyclophosphamide (CPA) / mL

The positive control compound, CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations.

In the first experimental series, the test item markedly reduced the mitotic index of the V79 cells at the concentration of 889 µg/mL. At higher concentrations no metaphases were present. In the second series, metaphases were found up to the concentration of 1580 µg/mL.

The test item did not lead to an increase in the number of polyploid cells. However, the substance significantly increased the proportion of cells with aberrant chromosomes as compared to the actual controls. This result was confirmed in the two independently performed experimental series.

In conclusion, the test item was clastogenic in this in vitro test system in the presence of S9 mix.

An additional test was performed without exogenous metabolizing system with the following conditions:

Exposure times: 5 hours (1st test series) 25 and 35 hous (2nd series)

Preparation times: 25 and 35 hours

Concentrations evaluated: 88.9, 281 and 889 µg/mL

Positive control: 500 µg Ethylmethansulfonat (EMS) / mL

The positive control compound EMS, induced the expected clear increase in the proportion of cells with chromosomal aberrations.

The test item precipitated in the culture medium at the highest concentration tested, i.e. 889 (µg/mL). A reduction in the mitotic index of the V79 cells was seen at the concentration of 889 µg/mL in the 1st experimental series.

Treatment of V79 cell cultures with the test item weakly increased the proportion of cells with aberrant chromosomes at two dose levels in one experimental series. These effects, however, were not seen in the first series performed. The test item showed equivocal effects of uncertain biological relevance in this in vitro test system in the absence of S9 mix (reference 7.6.1 -2).

In vivo Micronucleus test

A micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines and the OECD Guideline for Testing of Chemicals No. 474.

The substance was given once intraperitoneally by gavage to male rats, at doses of 100, 316 and 1000 mg/kg body weight. Rats of the negative control group received the solvent alone, i.e. an intraperitoneal dose of 10 mL Miglyoloil / kg body weight. The animals of the positive control group were treated with an oral dose of 16.5 mg cyclophosphamide/kg body weight.A total of 30 animals was used.

Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the highest dose groups (1000 mg/kg) preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the lower dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. For microscopic investigation one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined. The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micro-nucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.

The highest test material dose induced clear clinical signs of toxicity. No relevant treatment-related decrease in body weight (in comparison to the solvent control) occurred and no relevant variation was observed for the quotient normochromatic : polychromatic erythrocytes.

The mean numbers of polychromatic erythrocytes with micronuclei for the negative control (solvent) were all in or very close to the expected range predetermined by historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.

No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

The substance was not mutagenic in the micronucleus test in male rats under conditions where the positive control exerted potent mutagenic effects (reference 7.6.2 -1).

Justification for classification or non-classification

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.