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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:
experimental study
Adequacy of study:
key study
Study period:
08 Dec 1987 - 22 Feb 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: clear yellow liquid
- Analytical purity: no data
- Lot/batch No.: #K505-001-005
- Storage condition of test material: at room temperature in a clear glass bottle

Test animals

Species:
mouse
Strain:
other: CD-1 Albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, USA
- Age at study initiation:
Dose range finding study: 11 weeks
Main study: 7.5 weeks
- Weight at study initiation:
Dose range finding study: 32-40 g (males), 28-32 g (females)
Main study: 26-35 g (males), 24-29 g (females)
- Assigned to test groups randomly: yes, on following basis: body weight
- Housing: 5 animals of the same sex and dose group per cage in stainless steel wire mesh cages
- Diet: Rodent Lab Blox, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no.: #37F-0555
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test solutions were prepared prior to dosing by dissolving the test material in corn oil.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
single treatment
Post exposure period:
30, 48 and 72 h (dose groups and vehicle control)
30 h (positive control)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250, 500, 1000, 2000 and 5000 mg/kg bw
Basis:
actual ingested
Dose range finding study
Remarks:
Doses / Concentrations:
100, 250 and 500 mg/kg bw
Basis:
actual ingested
Main study
No. of animals per sex per dose:
Dose range finding study: 2
Main study: 5 (dose groups 100 and 250 mg/kg bw and control groups), 7 (dose group 500 mg/kg bw)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 60 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose range finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The time of sacrifice and cell harvest were determined from the time of dosing.

DETAILS OF SLIDE PREPARATION: Slides were fixed in absolute methanol and stained for 20 min in Giemsa-solution.

CODING OF SLIDES: Slides were coded randomly by study number and number designation.
Evaluation criteria:
As positive micronuclei were judged: uniform, darkly stained typically round (occasionally almond or tear drop shaped) bodies in the cytoplasm of PCE. Inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plane of the cell are judged to be artefacts. Cells containing more than one micronucleus are scored as one micronucleated PCE.
Statistics:
STATISTICS:
One tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE. The ratio of PCE/NCE was also calculated based on 1000 erythrocytes for each animal. The proportion of PCE per 1000 erythrocytes per animal was calculated by pairwise two-tailed t tests after an arc sine transformation was performed. Statistical significance was judged at p = 0.05 and p = 0.01 levels. All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs: In most animals decreased body tone and abnormal gait were observed.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 - 5000 mg/kg bw
- Clinical signs of toxicity in test animals: see Table 3 under 'Any other information on results incl. tables'.

RESULTS OF DEFINITIVE STUDY
- All animals at high dose level survived. Therefore, the extra animals were sacrificed but not included in the analysis.

Any other information on results incl. tables

Table 2. Toxic signs observed during the main study.

Exp group

Dose

Toxic signs

Vehicle control

(corn oil)

10 mL/kg bw

- no pharmacotoxic signs were observed

- no mortality occurred

Test substance

100 mg/kg bw

- at 5 h one female exhibited diarrhoea

- at 5, 24, 48 and 72 h one or two animals exhibited decreased body tone

Test substance

250 mg/kg bw

- all animals exhibited abnormal gait at all observation time points

- decreased body tone in several animals at most observation time points

Test substance

500 mg/kg bw

- all animals exhibited abnormal gait at all observation time points

- most animals exhibited decreased body tone at 5, 24, 48 and 72 h 

Positive control

(cyclophosphamide)

60 mg/kg bw

- no pharmacotoxic signs were observed

- no mortality occurred

Table 3. Toxic signs observed during the dose range finding study. 

Dose group

Toxic signs

250 mg/kg bw

- 2/2 females showed diarrhoea

- 2/2 males exhibited abnormal gait

4 h: 1/2 males exhibited decreased body tone and activity

24 h: 1/2 males was dead

24, 48 and 72 h: remaining animals showed abnormal gait and decreased body tone

500 mg/kg bw

24, 48 and 72 h: 4/4 animals showed abnormal gait

4, 24, 48 and 72 h: 4/4 animals showed decreased body tone

1000 mg/kg bw

- all animals exhibited abnormal gait at all observation time points

4 h: one or more animals showed decreased body tone and activity, body drop and diarrhoea

24 h: 1/2 males and females were dead

24, 48 and 72 h: one or more of the remaining animals exhibited abnormal gait, decreased body tone and/or vocalization on touch

2000 mg/kg bw

4 h: all animals exhibited abnormal gait, decreased body tone and activity and body drop

4 h: 1/2 females and 1/2 males showed diarrhoea

24 h: 1/2 males and 2/2 females were dead, 1/2 males showed abnormal gait and stance, body drop, decreased body tone and activity and chromaturia

48 h: remaining animal showed abnormal gait, decreased body tone and activity, tremors, chromaturia, body drop and ptosis

72 h: remaining animal (male) was dead

5000 mg/kg bw

4 h: 1/2 females and 1/2 males were dead, remaining animals showed decreased body tone and activity, body drop, abnormal gait, slight tremors and/or piloerection

24 h: all animals were dead

Table 4: Results of the in vivo micronucleus assay.

 

Mean

PCEs / 1000 NCEs

at sampling time

Total micronuclei

per 1000

PCEs at

sampling time

Exp group

Number

of animals

Dose [mg/kg]

30 h

48 h

72 h

30 h

48 h

72 h

Vehicle control

(corn oil)

10

10 mL/kg

1.865

2.089

1.901

20

17

718

Positive control

(cyclophosphamide)

10

60

1.565

n.d.

n.d.

187*

n.d.

n.d.

Test substance

10

100

2.068

1.870

1.910

11

17

21

Test substance

10

250

2.309

2.269

1.674

13

15

16

Test substance

10

500

2.664

1.914

2.149

12

20

20

n.d. = not determined; *statistically significant (p<0.01)

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative