Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No mutagenicity was observed in a valid Ames test in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 at doses of 5, 10, 25, 50, 100, 250 µg/plate.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No information on the purity of the substance and number of cells used. No individual plate counts, no statistical analyses. Only four strains tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 plus co-factors
Test concentrations with justification for top dose:
without microsomal activation : 10, 25, 50, 100, and 250 μg/0.1 mL
with microsomal activation: 5, 10, 25, 50, and 100 μg/0.1 mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Remarks:
with and without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3 out of 6 plates per dose with activation were preincubated

NUMBER OF REPLICATIONS:
In the experiments without the addition of microsomal activation mixture, three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group). In the experiments with activation mixture six Petri dishes were used per strain and per group; three of each set of six were pre-incubated

POSITIVE CONTROLS:
Without S9:
Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine, 2 µg/0.1 ml DMSO;
Strain TA 1537: 9(5)-aminoacridine hydrochloride monohydrate, 100 µg/0.1 ml DMSO
Strain TA 98: daunoblastin, 50 µg/0.1 ml DMSO;
Strain TA 100: 4-nitroquinoline-n-oxide, 10 µg/0.1 ml DMSO.
With S9:
Strain TA 100: cyclophosphamide, 100 and 250 µg/0.1 ml physiological saline
Evaluation criteria:
The test substance was considered to be non-mutagenic if the colony count in relation to the negative control was not doubled at any concentration.
Statistics:
When the colonies had been counted, the arithmetic mean was calculated.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation: at 100 and 250 μg/0.1 mL in soft agar
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1. Salmonella mutagenicity test: reverse mutation without microsomal activation. Numbers represent the arithmetic mean of histidine-prototrophic colonies.

Test substance Strain of S. typhi-murium used
TA 98 TA 100 TA 1535 TA 1537
control 28 115 11 8
10 μg/0.1 mL 24 118 11 8
25 μg/0.1 mL 24 151 14 5
50 μg/0.1 mL 24 149 17 6
100 μg/0.1 mL 24 136 13 7
250 μg/0.1 mL 24 163 16 7

Table 2. Salmonella/Mammalian microsome mutagenicity test: reverse mutation with microsomal activation. Numbers represent the arithmetic mean of histidine-prototrophic colonies.

n/n: first figures are values without, second figures values with pre-incubation.

Test substance Strain of S. typhi-murium used
TA 98 TA 100 TA 1535 TA 1537
control 18 / 21 173 / 165 18 / 20 8 / 7
5 μg/0.1 mL 23 / 25 179 / 202 17 / 17 9 / 9
10 μg/0.1 mL 22 / 25 181 / 188 21 / 20 8 / 5
25 μg/0.1 mL 23 / 21 190 / 217 26 / 20 7 / 8
50 μg/0.1 mL 23 / 23 177 / 189 21 / 19 7 / 7
100 μg/0.1 mL 23 / 22 157 / 155 17 / 18 7 / 7
Conclusions:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment revealed no significant differences.

Genetic toxicity in vivo

Description of key information

No genotoxicity was observed in a dominant lethal study in mice, in the chromosome aberration study in Chinese hamsters and in the micronucleus assay in Chinese hamsters. All studies are adequate regarding the reporting details and were performed with doses of up to 2000 or 3000 mg/kg body weight. The study design is adequate in comparison with the respective OECD testing guidelines, with the exception of the dominant lethal study which lacks positive control data.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Fa. A. THOMAE, D-Biberach a.d.Riss
- Weight at study initiation: 23-31 g (females), 24-33 g (males)
- Diet (e.g. ad libitum): NAFAG No.196
- Water (e.g. ad libitum): Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 55 ± 5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 500, 1000, and 2000 mg/kg in 20 mL/kg solution
- Amount of vehicle (if gavage or dermal): 5 % in 20 mL/kg solution
Duration of treatment / exposure:
2 days
Frequency of treatment:
daily
Post exposure period:
24 h after the second application the animals were sacrificed.
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 2 / 128 mg/kg
Tissues and cell types examined:
Bone marrow from both femurs
Details of tissue and slide preparation:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with 0.5 μL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. At the next day the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Grimwald solution/water 1/1 for 2 min subsequently with Giemsa solution 40% for 20 min. After having been rinsed i n methanol 55 % for 5-8 sec and subsequently with distilled water the air-dried slides were cleared in Xylol and mounted in Eukitt.
Evaluation criteria:
1000 bone marrow cells were scored from each animal and the following anomalies were registered:
a) Single Jolly bodies,
b) fragments of nuclei in erythrocytes ,
c) micronuclei in erythroblasts ,
d) micronuclei in leucopoietic cells,
e) polyploid cells
Statistics:
The significance of difference was assessed by χ2 -test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, the positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. Here the mean percentage of anomalies was 4.27, whereas the negative control yielded a percentage of 0.10. The difference is highly significant (p<0.05).

The effect of the test article and cyclophosphamide on bone marrow cells of Chinese hamster. Percent of cells with anomalies of nuclei


Treatment










Control Cyclophosphamide test substance

0.5 % CMC 64 mg/kg 500 mg/kg 1000 mg/kg 2000 mg/kg
Number and sex of animals
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
Single Jolly bodies 0.1 0.1

0.2 0.1 3.8 3.0 2.2 3.0 3.5 2.5
0.1 0.1 0.1 0.2

0.2 0.1
0.2

0.1 0.2 0.2

Fragments of nuclei in erythrocytes





0.4 0.5 0.3 0.8 0.8 0.4

















Micronuclei in erythroblasts





0.9 0.1 0.2 0.4 0.7 0.6

















Micronulclei in leucopoieetic cells


























0.1

Polyploid cells
0.1



0.5 0.3 0.1 0.2 0.3 0.1



0.1 0.2
0.2







0.1
Total 0.1 0.2 0.0 0.0 0.2 0.1 5.6 3.9 2.8 4.4 5.3 3.6 0.0 0.1 0.1 0.1 0.3 0.2 0.0 0.4 0.1 0.0 0.2 0.0 0.0 0.1 0.2 0.3 0.1 0.0
Conclusions:
It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro

No mutagenicity was observed in the Ames test in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 at doses of 5, 10, 25, 50, 100, 250 µg/plate both in the presence and absence of S9 prepared from Arochlor 1254 induced rats. The study design and the reporting details are adequate for hazard assessment. Precipitation was recorded at and above 100 µg/plate (-S9) in soft agar. A similar result was observed in a supporting study where the test material was reported to be negative with and without S9 mix at dose levels of up to 400 µg/plate in tester strains TA98, TA100, TA1535 and TA1537.

In vivo

No genotoxicity was observed in a dominant lethal study in mice, in the chromosome aberration study in Chinese hamsters and in the micronucleus assay in Chinese hamsters. All studies are adequate regarding the reporting details. In design, the micronucleus study is performed comparable to OECD testing guideline 474 and with doses of 500, 1000 and 2000 mg/kg body weight; therefore, it is chosen as key study. In the micronucleus study, three male and three female hamsters were treated by gavage and sacrificed 24 h after the second application. From each animal, 1000 bone marrow cells were scored. As no gender specificity is known for this substance, the use of 3 males and 3 females is justified. Toxicity symptoms were not recorded which is in line with the overall low toxicity of the substance. The applied doses were at the limit dose level. The following anomalies were recorded: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploid cells. The positive control had the expected response. For the chromosome aberration study, which was performed with doses of 500, 1000 and 2000 mg/kg body weight, a total of four instead of five animals were evaluated. This is the only deviation from OECD testing guideline 475. Six male and four female hamsters were treated by gavage and sacrificed 6 h after the second application. From the bone marrow, chromosome preparations were made and slides of 2 animals per sex and group were scored (100 metaphases per animal). No aberrations were found in the treated groups relative to the negative control. The positive control had the expected response. For the dominant lethal study, no positive control data are available. This study was performed with a single application of 1000 and 3000 mg/kg body weight to NMRI mice. Its design is comparable to OECD testing guideline 478. The mating success was 89.6%, 89.2% and 93.8% at control, 1000 and 3000 mg/kg, respectively. No effects at any time point was observed for the mean number of implantation sites per pregnant female, mean number of live fetuses per litter and the mean number of embryonic deaths per female. The duration of the test is sufficient to cover the complete spermatogenesis. The substance was found to be non-carcinogenic in rats and mice.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.