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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-study performed under GLP and QAU

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(only 1000 immature erythrocytes/animal were scored)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
CIBA-Geigy Limited, Basel, Switzerland
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: red powder

Test animals

Species:
mouse
Strain:
other: MAGf
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: in-house breeding
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 24-31g (females), 31-40 g(males)
- Assigned to test groups randomly: yes (random selection with the aid of a computer generated random list)
- Fasting period before study: 12 hours
- Housing: individually or in groups of two
- Diet (ad libitum): NAFAG No 890 Tox
- Water (ad libitum): drinking water
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-88
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
A solubility test was performed to determine the highest applicable dose level (suspension) of the test substance for the tolerability test (up to a top dose level of 5000 mg/kg body weight). Carboxymethylcellulose (CMC), 0.5% in water was found to be the best suited vehicle, yielding an applicable suspension at the highest dose level of 5000 mg/kg.
- Amount of vehicle (if gavage or dermal): 20 ml/kg bw
Frequency of treatment:
Single treatment
Post exposure period:
5000 mg/kg bw dose group and negative control group: 24 and 48 hours
1250 and 2500 mg/kg bw dose group and positive control: 24 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, dissolved in bidistilled water
- Route of administration: orally (The oral route was chosen because it provides the best experimental model for the expected exposure route in man)
- Doses / concentrations: 64 mg/kg

Examinations

Tissues and cell types examined:
femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high dose applied (limit dose, 5000 mg/kg bw) was toxic as manifested in a range finding test by various symptoms.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): From the high dose group and from the negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed 24 hours after application. The animals were sacrificed by CO2 gas. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei: Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Thereof smears were made. They were air-dried and then stained with May-Grünwald/Giemsa solution. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted.

DETAILS OF SLIDE PREPARATION: 1000 cells/animal scored

Criteria for scoring micronuclei:
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and were not scored as micronuclei. Cells containing more than one micronucleus are only counted once.
Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good differentiation between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 1000 polychromatic erythrocytes (PCE), and the ratio of PCE to normochromatic erythrocytes (NCE) among a total of 1000 erythrocytes was determined for each slide.
Evaluation criteria:
Assay evaluation criteria:
The results of the experiments were evaluated with respect to the mean number of PCEs with micronuclei. The groups compared differed by treatment, sampling time and sex of the animals. Since there was no significant difference between animals of either sex, the data from females and males were pooled for evaluation.
Criteria for a positive effect:
The test substance is considered to be active in this test system if at any group treated with the test substance the mean number of micronucleated PCEs exceeds the value of 0.20% and if there is a statistically significant difference of the number of micronucleated PCEs in comparison with the negative control.
Exceptions:
If the positive effect occurs in a minority of the treated animals only and if their number of micronucleated normochromatic erythrocytes (NCEs) is also enhanced in comparison with the respective negative control, the effect on PCEs is not attributed to the treatment. If the limits of the criteria for a positive or for a negative response are reached, the Study Director additionally interpreted the results based on previous experience with this test system.
Assay acceptance criteria:
1. The result of the experiment should not be influenced by a significant technical error or a recognized artifact.
2. The high dose of the test substance applied should be the maximum tolerated dose causing no death in a group of four animals in the range finding-test. In the absence of lethality in the tolerability test, the high dose should be up to a maximum of 5000 mg/kg body weight or the highest applicable dose due to the limited solubility of the test substance.
3. The quality of the slides must allow a clear differentiation between PCEs and NCEs.
4. The mean number of micronucleated PCEs in the negative control groups should not exceed the value of 0.20%.
5. The positive control should fullfill the criteria for an active substance.
Statistics:
The significance of differences was assessed by the Chi-Squared-Contingency-Test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
pale red skin was observed in all animals dosed with 5000 mg/kg bw and weight of loss in one male of the 2500 mg/kg bw dose group (same effects were observed in the range -finding study, see "additional information on results")
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose: 5000 mg/kg
- Clinical signs of toxicity in test animals: transient loss of weight, ataxia, ventral recumbency, unkempt fur, pale red skin and red feces
No further investigations.

Any other information on results incl. tables

Table 1: Micronuclei formation in mouse bone marrow cells of animals treated 24 hours

Treatment

Sex

PCEs/1000 erythrocytes (mean)

Ration PCE/NCE

Micronucleated PCEs found in 5000 PCEs #

% of micronucleated PCEs

Neg. Control (0.5% CMC)

Males

414

0.71

1

0.02

 

Females

460

0.85

1

0.02

 

Pooled data

 

 

2

 

1250 mg/kg

Males

447

0.81

1

0.02

 

Females

443

0.79

7

0.14*

 

Pooled data

 

 

8

0.08

2500 mg/kg

Males

453

0.83

1

0.02

 

Females

452

0.83

3

0.06

 

Pooled data

 

 

4

0.04

5000 mg/kg

Males

411

0.70

3

0.06

 

Females

438

0.78

2

0.04

 

Pooled data

 

 

5

0.05

Pos. Control (CP 64 mg/kg)

Males

377

0.60

91

1.82*

 

Females

442

0.79

42

0.84*

 

Pooled data

 

 

133

1.33*

# = in a total number of 5 animals/sex

CMC =Carboxymethylcellulose

CP = Cyclophosphamide

* = Number of micronucleated PCEs statistically significant different from negative control (Level of significance p=0.05)

Table 1: Micronuclei formation in mouse bone marrow cells of animals treated 48 hours

Treatment

Sex

PCEs/1000 erythrocytes (mean)

Ration PCE/NCE

Micronucleated PCEs found in 5000 PCEs #

% of micronucleated PCEs

Neg. Control (0.5% CMC)

Males

452

0.83

3

0.06

 

Females

446

0.81

2

0.04

 

Pooled data

 

 

5

0.05

5000 mg/kg

Males

450

0.82

3

0.06

 

Females

478

0.92

4

0.08

 

Pooled data

 

 

7

0.07

CMC =Carboxymethylcellulose

At all three dosage groups and at both sampling times, with one exception (females, 1250 mg/kg, 24 hours), there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the test substance as compared with the respective negative control animals. In the group of females dosed with 1250 mg/kg (24 hours sampling time) there was a very weak, but statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the test substance. No such increase was observed in the corresponding group of males and there was no dose-dependency in the groups of females. The mean percentage of micronucleated PCEs (0.14%) did not meet the criteria for a positive response and was within the range of the negative historical control. Therefore, the slight increase in the number of micronucleated polychromatic erythrocytes is attributed to spontaneously occurring events and is considered to be of no biological relevance.

At the 24 hours sampling time in the groups treated with the test substance the mean percentage of micronucleated PCEs was 0.08 (1250 mg/kg), 0.04 (2500 mg/kg) and 0.05 (5000 mg/kg) respectively. The respective negative control value for this group was 0.02%.

At the 48 hours sampling time in the group treated with 5000 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.07 compared to a negative control value of 0.05.

In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 1.33.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative