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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The substance was not mutagenic in the Ames test in Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, tested both in the absence and presence of a metabolic activation system (Ciba-Geigy, 1984). No genotoxicity was observed in Chinese hamsters at doses of 1000, 2000 and 4000 mg/kg bw in the nucleus anomaly test (Ciba-Geigy, 1985). The test article was negative in an HPRT Study with and without metabolic activation (BASF, 2013)
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:
January 7, 1985 - May 6, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP study with sufficient details acceptable for assessment.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1000 instead of 2000 cells scored per animal
Principles of method if other than guideline:
Treatment consisted of one daily dose (gavage) of 1000, 2000 or 4000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were analyzed for nucleus anomalies resulting from chromosomal damage.
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
other: random outbred
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln.
- Weight at study initiation: females: 20-34 g, males: 22-34 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Diet (e.g. ad libitum): Standard diet; NAFAG No.924.
- Water: Tap water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 42-44
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 0.5% aqueous solution of sodium carboxymethylcellulose (CMC)
- Amount of vehicle (if gavage or dermal): 20 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: No details.
Duration of treatment / exposure:
Treatment consisted of one daily dose on each of two consecutive days.
Frequency of treatment:
Once/day
Post exposure period:
24 h after the second application, the animals were sacrificed by dislocation of the cervical vertebrae.
Remarks:
Doses / Concentrations:
1000, 2000, 4000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
- In the tolerability test: 2
- in the mutagenicity test: 6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral/gavage
- Doses / concentrations: 128 mg/kg in 20 ml/kg bw 0.5% CMC
Tissues and cell types examined:
Bone marrow was harvested from the shafts bf both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test substance to be applied in the mutagenicity assay. In this experiment the dose of 4000 mg/kg was determined as the highest applicable in the mutagenicity assay,, together with further two doses, diminishing by a factor of 0.5.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts bf both femurs. In a sicliconized pipette filled with approx. 0.5 µl rat serum the bone marrow was drawn up. In order to receive a homogenous 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 polishedcover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted in Eukitt.
Evaluation criteria:
The slides of three female and three male animals each of the negative control group, the positive control group and of the groups treated with various doses were examined. 1000 bone marrow cells each were scored per 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 chi-square test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
One male animal of the 4000 mg/kg-dose group died in the course of the experiment.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
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 8.12, whereas the negative control yielded a percentage of 0.03. The difference is highly significant (p< 0.05),

Table 1: Percent of cells with anomalies of nuclei, 24 h after second application of test substance.

 

Number of animal

Sex of animals

Single Jolly-Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythroblasts

Micronuclei in leucopoietic cells

Polyploid cells

Totals   

Vehicle control

1

F

0.0

 

2

F

0.0

 

3

F

0.0

 

4

M

 0.2

 

0.2

 

5

M

0.0

 

6

M

0.0

Cyclophosphamide, 128 mg/kg bw

1

F

3.7

1.4

1.8

0.1

7.0

 

2

F

6.8

1.2

0.8

8.8

 

3

F

5.1

2.3

1.9

0.1

9.4

 

4

M

8.6

1.2

2.0

0.3

12.1

 

5

M

4.4

0.7

1.1

0.1

6.3

 

6

M

3.7

0.7

0.3

0.3

0.1

5.1

Test substance, 1000 mg/kg bw

1

F

0.1

 

0.1

 

2

F

 

0.0 

 

3

F

 

0.0

 

4

M

0.1

0.0

 

5

M

 

0.0

 

6

M

 

0.0

Test substance, 2000 mg/kg bw  1  F          0.1
   2  F  0.1        0.1
   3  F          0.0
   4  M          0.0
   5  M          0.0
   6  M  0.1        0.1
 Test substance, 4000 mg/kg bw  1  F  0.1        0.1
   2  F  0.2        0.2
   3  F  0.2        0.2
   4  M  0.2        0.2
   5  M  0.1        0.1
   6  M  0.1      0.1
Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this experiment, no evidence of mutagenic effects were noted in Chinese hamsters treated with the test substance.
Executive summary:

The test substance was evaluated for mutagenicity in a Micronucleus test in Chinese hamsters. Treatment consisted of one daily dose of 1000, 2000 or 4000 mg/kg on each of two consecutive days. The test substance was applied orally by stomach tube. The animals were sacrificed 24 h after the second application and bone marrow smears prepared. The bone marrow smears from animals treated with the various doses of the test substance showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 8.12% cells with anomalies of nuclei. This is significantly different from the controls (0.03%) treated with the vehicle (0.5% CMC) alone. Therefore, the test substance was considered to be not mutagenic under the presented experimental conditions.

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

Additional information

Additional information from genetic toxicity in vivo:

The genotoxicity was investigated in bacterial mutagenicity test (Ciba-Geigy, 1984), in an HPRT test (BASF, 2013) and in a mammalian erythrocyte micronucleus test (Ciba-Geigy, 1985). None of the studies gave indication of genotoxic properties.

Ames test

The test substance was not mutagenic in the Ames test in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, tested both in the absence and presence of S9 mix prepared from Arochlor-1254 -induced rats following the procedure described by Ames (Ames et al., 1973 - 1975). The study was not performed under GLP and the test article was not tested with bacterial strains which have an AT base pair at the primary reversion site (E.coli WP2 or S. typhimurium TA102). However, the study contains sufficient details, is well documented and includes a QAU statement suggesting GLP-like conditions. Incubations were performed in a plate-incorporation design with an independent repeat experiment. Tested concentrations were up to 5000 μg per plate, with precipitation occurring at concentrations from 1280 μg/plate and above. Acetone was used as vehicle. Cytotoxicity was observed from 1280 µg/plate, less pronounced in experiments with S9 mix. The validity of the experiment was confirmed by incubation with positive control substances. Treatment with the test item did not cause an increase in the number of reverse mutations, thus the test article is considered to be not mutagenic in the Ames test.

HPRT Study

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The test item was dissolved in DMSO. The concentration range of the main experiments (0.7 – 44.0 µg/ml) was limited by cytotoxic effects. No precipitation was noted up to the maximum evaluated concentration with and without metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 11.0 μg/mL without metabolic activation and 44.0 μg/mL with metabolic activation. In the second experiment no relevant cytotoxic effects occurred up to the maximum analysable concentration of 44.0 μg/mL. Exceedingly severe cytotoxicity precluded analysis at the next higher concentration of 88.0 μg/mL. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency exceeded the threshold of three times the mutation frequency of the solvent control in the second culture of experiment II at 44.0 μg/mL without metabolic activation and in the first culture of experiment II at 5.5 and 44.0 μg/mL with metabolic activation. However, the increases were judged as biologically irrelevant as they were based on the rather low corresponding solvent control of just 8.4 and 6.7 mutant colonies per 106 cells. The absolute values of the mutation frequency remained well within the historical range of solvent controls. Furthermore, the parallel cultures showed no comparable increase under identical experimental conditions. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore, considered to be non-mutagenic in this HPRT assay.

Genetic toxicity in vivo

The genotoxic potential of the test substance was assessed in a nucleus anomaly test in Chinese hamsters. Although not performed under GLP, the study was performed under GLP-like quality control with QAU surveillance and contains sufficient documentation. The test substance in 0.5 % CMC was administered by gavage to 6 male and female Chinese hamsters per dose group. Treatment consisted of one daily dose of 1000, 2000 and 4000 mg/kg bw on each of two consecutive days. Cyclophosphamide given at 128 mg/kg bw served as positive control, a negative control group animals received the vehicle only. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made and interphase cells were observed for anomalies. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 8.12 % cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test article.


Justification for selection of genetic toxicity endpoint
GLP-compliant guideline study in vivo

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC.

 

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.