2-Isobutyryl-3,N-diphenylacrylamide

Genetic toxicity: in vivo

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:

1997-03-18 to 1999-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 92/69/EWG, B.12 (Mikrokerntest); OECD 474

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): PD 132408
- Substance type: white powder
- Physical state: solid
- Storage condition of test material: Roomtemperature in the dark

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Weight on despatch: males weighed between 24 and 28 grams, females weighed between 18 and 22 grams
- Assigned to test groups randomly: yes
- Diet: food and water ad libitum
- Acclimation period: min 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 50%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil (Diss Office Line, Batch # F8048 for PD 132408
water for positve control Mitomycin C

Details on exposure:

All animals in all groups were dosed with the standard volume of 20 mL/kg.

Duration of treatment / exposure:

Single treatment

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for five animals in all sex/dose/substance groups
48 hours for five animals of both sexes in the control and in the high dose group (2000 mg/kg)

No. of animals per sex per dose:

Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

five animals of both sexes were treatet with mitomycin c (12 mg/kg bodyweight)

Examinations

Tissues and cell types examined:

immature erythrocytes from bone marrow of both femurs

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose selection was based on a preliminary toxicity test with each two animals of both sexes in four dose groups ( 250, 500, 1000, 2000 mg/kg bodyweight)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
see above


DETAILS OF SLIDE PREPARATION:
The femurs were cleared from tissue and the proximal epiphysis removed from each bone. The bone marro of both femurs from each animal was flushed out and poolded in a total volume of 2 mL of prefiltered foetal calf serum using a 2 mL disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976) At least three smears were made from each animal. The prepared smears were fixed in methanol (>10 min). After air dryining the smears were stained for 10 minutes in 10% Giemsa prepared by 1:9 dilution of Gurrs improved R66 Giemsa (BDH) with purified water. Following rinsing in purified water adn differentiation in buffered purified water, the smears were air-dried and mounted with coverslips using DPX.


METHOD OF ANALYSIS:


OTHER:

Evaluation criteria:

The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 immature erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in case of technical problems with the first smear.
A positive response is normally indicated vy a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (p<0.01). Furthermore individual and /or group mean values should exceed the laboratory historical control range.
Bone marrow cell toxicity is normally indicated by a substantial statistically significant dose-related decrease in the proportion of immature erythrocytes (p<0.01) This decrease would normally be evident at the 48 houzr sampling time.

Statistics:

One-sided p-values by permutation; Linear by Linear Association test for trend in a step-down fashion if significance is detected; for individual inter-group comparisons straight forward permutation test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with PD 132408 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).
The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.
Slight signs of toxicity were observed in the highest dose group ( 2000 mg/kg): Underactivity, Fasiculations
The positive control compound, mitomycin C, produced large, highly significant (p<0.001) increases in the frequency of micronucleated immature erythocytes.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that PD 132 408 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by intragestic gavage.

Executive summary:

The substance PD 132408 was examined in vivo for clastogenic effects in bone marrow cells of mice. There was no significant induction of micronucleated immature erythrocytes after treatment by ingastric gavage.