Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test: negative (reference 7.6.1 -1).

Chromosome aberration test in vitro: negative (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:
1990-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
there was no strain tested with main DNA target AT (trpE gene) in the first study. Two other studies were conducted with E.coli only.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
there was no strain tested with main DNA target AT (trpE gene) in the first study. Two other studies were conducted with E.coli only.
Principles of method if other than guideline:
there was no strain tested with main DNA target AT (trpE gene) in the first study. Two other studies were conducted with E.coli only.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor-induced male rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50, 250, 500, 1000, 2000, 3000, 4000, 5000, 7500, 10000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 250, 500, 1000, 2000, 3000, 4000, 5000, 7500, 10000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
methylmethanesulfonate
other: 2-Aminoanthracene; Daunomycin;
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 1250 µg/plate
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 1538
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
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
at precipitating concentrations only; Batch No. 315
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
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
at precipitating concentrations only; Batch No. 315
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
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Batch No. HE 227/92
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
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Batch No. HE 227/92
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

Please refer to the attached background material.

The test item dose-dependently increased the number of revertants of both, E. coli WP2 and WP2 uvrA. The effects were reproduced in the repeat experiments performed. Maximal increases of 3- to 5-fold over the respective solvent controls were induced. These

effects however occurred at high test material concentrations which strongly precipitated on the agar plates.

This weak mutagenic effects were re-produced in another independent study conducted with the test item in E. coli strains. However, testing a purified batch of the test item negative results were obtained. Therefore, the weak mutagenicity observed is highly likely related to an impurity and not to the test item itself.

Conclusions:
Under the conditions of this study, the test item was not mutagenic in S. typhimurium strains but weakly mutagenic in E. coli.
Executive summary:

Mutagenic potential of the test item was investigated using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used. The test item was tested in two series of experiments at the following concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

In another study, mutagenic potential of the test item was investigated using Escherichia coli WP2 and WP2 uvrA as tester strains. Again, the plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used. The test item was tested in four series of experiments at the following concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

A third study was conducted using Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 and Escherichia coli WP2 and WP2 uvrA as tester strains. The plate incorporation test with and without addition of liver S9-Mix from Aroclor 1254-pretreated rats was used. The test item was tested in two series of experiments at the following concentrations: 25, 50, 250 1250, 2500, 5000, and 10000 µg/plate.

9-Aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2 -phenylene diamine, and sodium azide served as positive control compounds for testing the bacteria in all three studies. 2-Aminoanthracene was used for testing the bacteria and the activity of the S-9 preparation. The positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used. Thus, all three studies are considered to be valid.

With and without addition of S-9 as the metabolizing system, did not show any mutagenic activity in the Salmonella typhimurium strains at the concentration range used.

The test item dose-dependently increased the number of revertants of both, E. coli WP2 and WP2 uvrA. The effects were reproduced in the repeated experiments performed. Maximal increases of 3- to 5-fold over the respective solvent controls were induced. These effects however occurred at high test material concentrations which strongly precipitated on the agar plates.
In the third study conducted, this weak mutagenicity in E. coli strains was confirmed. However, with and without addition of S9-Mix as the external metabolizing system, no increases in the number of revertants over the respective solvent controls occurred with the purified test material. Thus, the test item was not mutagenic under the experimental conditions described. It is therefore very likely that chemical impurities are responsible for the mutagenic effects seen with the earlier tested batch.

In conclusion, the test item was weakly mutagenic in Escherichia coli WP2 and WP2 uvrA. Since the effects occurred at precipitating test material concentrations, it should however be noted that not the test item itself but, more likely, impurities may be responsible for the weak mutagenic effects found in this finding. This is confirmed by negative findings obtained with a purified batch tested.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-07-24
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Remarks:
Only executive summary available
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from aroclor 1254 induced rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation; 20 hour harvest): 125, 250, 500 µg/mL (female); 250, 400, 500 µg/mL (male)
Concentration range in the main test (with metabolic activation; 44 hour harvest): 500 µg/mL (male and female)

Concentration range in the main test (without metabolic activation; 20 hour harvest): 150, 300, 500 µg/mL
Concentration range in the main test (without metabolic activation; 44 hour harvest): 500 µg/mL
Vehicle / solvent:
Dimethylsulfoxid
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours
Fixation time:
1. Serie: 20 h

2. Serie: 20, 44 h
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see "overall remarks"
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the experimental conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes.
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce chromosome aberrations in cultured human lymphocytes. The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5 % CO2 / 95 % air, for 48 hours.

First experiment:
Lymphocyte cultures were exposed to the test or control substances, with or without S9 mix, for three hours and then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block, cells at the metaphase-stage of mitosis. As this first experiment was negative, the study was continued with a second experiment.

Second experiment:

- without S9 mix, cells were exposed continuously to the test or control substances.

- with S9 mix, cells were exposed to the test or control substances for three hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. For both experiments, after hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test substance was dissolved in dimethylsulfoxide (DMSO). The concentration-levels of the test item were as follows (two cultures/dose-level):

First experiment:

for treatment: 1.5, 5, 15, 50, 150, 500 µg/mL with and without S9 mix. In the absence of cytotoxicity at 150 µg/mL, this assay was used to choose the concentration-levels of a second assay without S9 mix: 75, 150, 300 and 500 µg/mL.

for chromosome aberrations scoring:

- without S9 mix: 150, 300, 500 µg/mL

- with S9 mix: 50, 150, 500 µg/mL

500 µg/mL being the lowest precipitating dose.

Second experiment:

for treatment:

- without S9 mix: 37.5, 75, 150, 300, 500 µg/mL for both harvest times,

- with S9 mix: 125, 250, 500 µg/mL for both harvest times plus 400 µg/mL for the male only.

for chromosome aberrations scoring:

- without S9 mix: 20-hour harvest: 150, 300,500 µg/mL

44-hour harvest: 500 µg/mL

- with S9 mix: 20-hour harvest: 125, 250, 500 µg/mL for the female

250, 400, 500 µg/mL for the male

44-hour harvest: 500 µg/mL for the male and the female.

The dose-levels of the positive controls were as follows:

without S9 mix, mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

with S9 mix, cyclophosphamide: 50 µg/mL.

 

The frequency of cells with structural chromosome aberrations in the vehicle and positive controls was as specified in the acceptance criteria and within the range of our historical data. The test substance did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with or without S9 mix, for both experiments and for the two harvest times. Under the experimental conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes.

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

Genetic toxicity in vivo

Description of key information

Micronucleus in vivo: Negative (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:
1990-05-30 to 1990-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: approx. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, approx. 18 hrs before treatment
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity: 30-70 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light
Route of administration:
oral: gavage
Vehicle:
Polyethylenglycol 400
Duration of treatment / exposure:
single gavage application
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
24 hours post-exposure period
Dose / conc.:
670 mg/kg bw/day (actual dose received)
Remarks:
24 hours post-exposure period
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
24 and 48 hours post-exposure period
No. of animals per sex per dose:
5 per sex per dose per post exposure period
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control: as recommended by the guideline
- Route of administration: oral (gavage)
- Doses / concentrations: 20 and 40 mg/kg bw (two groups)
Tissues and cell types examined:
bone marrow of femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: according to guideline the MTD was applied as high dose

TREATMENT AND SAMPLING TIMES: single treatment followed by either 24 or 48 hours post-treatment period

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt, F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.).
At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 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 same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points. A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the non-parametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Doses producing toxicity: at 2000 mg/kg bw unspecific signs of toxicity were observed but no mortality occurred; slight cytotoxicity (PCE/NCE) was seen at this dose level at 48 h post-treatment evaluation

Observations:
No increase in the micronucleus rate in animals treated with the test item.

Please refer to background material attached.

Conclusions:
Under the conditions of this study the test item was found to be not clastogenic.
Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single application of the test article the one 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 (PCE) per animal were scored. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw

In a pre-experiment 2000 mg/kg bw was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the test item had weak cytotoxic effectiveness. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used. 20 mg/kg bw cyclophosphamide administered per gavage was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

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

Additional information

Bacterial reverse mutation assay (Ames Test), reference 7.6.1 -1

Mutagenic potential of the test item was investigated using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used. The test item was tested in two series of experiments at the following concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

In another study, mutagenic potential of the test item was investigated using Escherichia coli WP2 and WP2 uvrA as tester strains. Again, the plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used. The test item was tested in four series of experiments at the following concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

A third study was conducted using Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 and Escherichia coli WP2 and WP2 uvrA as tester strains. The plate incorporation test with and without addition of liver S9-Mix from Aroclor 1254-pretreated rats was used. The test item was tested in two series of experiments at the following concentrations: 25, 50, 250 1250, 2500, 5000, and 10000 µg/plate.

9-Aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2 -phenylene diamine, and sodium azide served as positive control compounds for testing the bacteria in all three studies. 2-Aminoanthracene was used for testing the bacteria and the activity of the S-9 preparation. The positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used. Thus, all three studies are considered to be valid.

With and without addition of S-9 as the metabolizing system, did not show any mutagenic activity in the Salmonella typhimurium strains at the concentration range used.

The test item dose-dependently increased the number of revertants of both, E. coli WP2 and WP2 uvrA. The effects were reproduced in the repeated experiments performed. Maximal increases of 3- to 5-fold over the respective solvent controls were induced. These effects however occurred at high test material concentrations which strongly precipitated on the agar plates.
In the third study conducted, this weak mutagenicity in E. coli strains was confirmed. However, with and without addition of S9-Mix as the external metabolizing system, no increases in the number of revertants over the respective solvent controls occurred with the purified test material. Thus, the test item was not mutagenic under the experimental conditions described. It is therefore very likely that chemical impurities are responsible for the mutagenic effects seen with the earlier tested batch.

In conclusion, the test item was weakly mutagenic in Escherichia coli WP2 and WP2 uvrA. Since the effects occurred at precipitating test material concentrations, it should however be noted that not the test item itself but, more likely, impurities may be responsible for the weak mutagenic effects found in this finding. This is confirmed by negative findings obtained with a purified batch tested.

In vitro mammalian chromosome aberration test, reference 7.6.1 -2

The objective of this study was to evaluate the potential of the test substance to induce chromosome aberrations in cultured human lymphocytes. The test substance was tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. No preliminary cytotoxicity test was performed. Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosome aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index (MI). For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemogglutinin) and incubated at 37°C in a humidified atmosphere of 5 % CO2 / 95 % air, for 48 hours.

First experiment:
Lymphocyte cultures were exposed to the test or control substances, with or without S9 mix, for three hours and then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block, cells at the metaphase-stage of mitosis. As this first experiment was negative, the study was continued with a second experiment.

Second experiment:

- without S9 mix, cells were exposed continuously to the test or control substances.

- with S9 mix, cells were exposed to the test or control substances for three hours and then rinsed.

Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. For both experiments, after hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test substance was dissolved in dimethylsulfoxide (DMSO). The concentration-levels of the test item were as follows (two cultures/dose-level):

First experiment:

for treatment: 1.5, 5, 15, 50, 150, 500 µg/mL with and without S9 mix. In the absence of cytotoxicity at 150 µg/mL, this assay was used to choose the concentration-levels of a second assay without S9 mix: 75, 150, 300 and 500 µg/mL.

for chromosome aberrations scoring:

- without S9 mix: 150, 300, 500 µg/mL

- with S9 mix: 50, 150, 500 µg/mL

500 µg/mL being the lowest precipitating dose.

Second experiment:

for treatment:

- without S9 mix: 37.5, 75, 150, 300, 500 µg/mL for both harvest times,

- with S9 mix: 125, 250, 500 µg/mL for both harvest times plus 400 µg/mL for the male only.

for chromosome aberrations scoring:

- without S9 mix: 20-hour harvest: 150, 300,500 µg/mL

44-hour harvest: 500 µg/mL

- with S9 mix: 20-hour harvest: 125, 250, 500 µg/mL for the female

250, 400, 500 µg/mL for the male

44-hour harvest: 500 µg/mL for the male and the female.

The dose-levels of the positive controls were as follows:

without S9 mix, mitomycin C: 3 µg/mL (3 hours of treatment) or 0.2 µg/mL (continuous treatment),

with S9 mix, cyclophosphamide: 50 µg/mL.

 

The frequency of cells with structural chromosome aberrations in the vehicle and positive controls was as specified in the acceptance criteria and within the range of our historical data. The test substance did not induce any significant increase in the frequency of cells with structural chromosome aberrations, with or without S9 mix, for both experiments and for the two harvest times. Under the experimental conditions, the test substance did not induce chromosome aberrations in cultured human lymphocytes.

In vivo Micronucleus test, reference 7.6.2 -1

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single application of the test article the one 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 (PCE) per animal were scored. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE. The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw

In a pre-experiment 2000 mg/kg bw was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the test item had weak cytotoxic effectiveness. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used. 20 mg/kg bw cyclophosphamide administered per gavage was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data the test item does not require classification as genotoxic according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.