Benzonitrile, 3,3'-(2,3,5,6-tetrahydro-3,6-dioxopyrrolo[3,4-c]pyrrole-1,4-diyl)bis-

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:

1993-09-03 to 1994-01-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted may 26, 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

412-640-1

EC Name:

-

Cas Number:

84632-50-8

Molecular formula:

C20 H10 N4 O2

IUPAC Name:

3-[4-(3-cyanophenyl)-3,6-dioxo-2H,3H,5H,6H-pyrrolo[3,4-c]pyrrol-1-yl]benzonitrile

Details on test material:

- Analytical purity: 97%
- Appearance: orange powder

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 U.K. Limited, Margate, Kent, England
- Age at study initiation: approximately 35 days (on despatch)
- Weight at study initiation: 22 to 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: yes (overnight prior to and for two hours after oral dosing)
- Housing: groups of five in plastic disposable cages, sexes separated
- Diet: pelleted Biosure LAD 1 rodent diet ad libitum
- Water: tap water ad libitum
- Acclimation period: approximately 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 23°C
- Humidity: 44-58%
- Air changes: approximately 30 per hour
- Photoperiod: 12 hours artificial light per day

IN-LIFE DATES: From: 1993-09-28 To: 1993-10-22

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: methyl cellulose (MC)
- Concentration of test material in vehicle: 250 mg / mL
- Amount of vehicle: 20 mL / kg bodyweight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test item were prepared in aqueous 1 % methylcellulose on the morning of the test.

Duration of treatment / exposure:

not applicable (single oral gavage treatment)

Frequency of treatment:

once

Post exposure period:

negative control and test substance groups: sacrificed 24, 48 or 72 hours after dosing (5 males and 5 females each)
positive control group: sacrificed 24 hours after dosing

Doses / concentrations

Remarks:

Doses / Concentrations:
0 or 5000 mg / kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Preliminary experiment: 2 males and 2 females
Main study: 5 males and 5 females per time point of sacrifice

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: oral gavage
- Doses / concentrations: solution in 0.9% saline at a concentration of 0.6 mg/mL (prepared just prior to administration)

Examinations

Tissues and cell types examined:

- polychromatic erythrocytes
- normochromatic erythrocytes

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation and both femurs dissected out from each animal. A direct bone marrow smear was made onto a slide after dilution of the marrow with a drop of foetal calf serum. Smears, made from each femur, were fixed in methanol, air-dried and stained with Giemsa.

ANALYSIS:
- micronucleated cells per 1000 polychromatic erythrocytes per animal
- ratio polychromatic vs normochromatic cells

Evaluation criteria:

A positive response is normally indicated by a substantial, statistically significant increase (P<0.01) in the incidence of micronucleated polychromatic erythrocytes compared to the incidence for the concurrent vehicle control group for at least one of the sampling times; individual and/or group mean values should exceed the laboratory historical control range. A negative result is indicated when individual and group mean incidences of micronucleated polychromatic erythrocytes for animals treated with the test substance are not significantly greater than incidences for the concurrent control group and where these values fall within the historical control range. An equivocal response is obtained when the results cannot be adequately classified using the criteria for a positive or negative response.

Statistics:

Wilcoxon's sum of ranks (Non-parametric statistical methods, based on rank). Unless there is a substantial difference in response between sexes, results for the two sexes are combined to facilitate interpretation and maximise the power of statistical analysis.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but limit dose of 5000 mg / kg bw tested

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: limit dose (5000 mg / kg bw)
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analyzed: not analysed
- Rationale for exposure: limit dose
- Observation period: 72 hours

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no
- Ratio of PCE/NCE: unaltered
- Appropriateness of dose levels and route: adequate
- Statistical evaluation: no significances

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative