Registration Dossier

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:
From May 2, 1988 to March 6, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented, according to accepted guideline, with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
hamster, Chinese
Strain:
other: Chinese
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation:
a) Tolerability test: 27-35 g (males and females).
b) Mutagenicity test: 22-35 g (males); 21-34 g (females).
- Assigned to test groups randomly: Yes.
- Housing: Individually housed.
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: At least 3 days.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23°C.
- Humidity (%): 43-54%.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 0.5%
- Amount of vehicle (if gavage or dermal): 20 mL/kg.
- Manufacturer of Carboxymethyl cellulose (CMC): Hercules Comp.,USA.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 4000 mg/kg in 20 mL/kg CMC 0.5%.


DIET PREPARATION: Standard diet: NAFAG No. 924.
Duration of treatment / exposure:
Single exposure.
Frequency of treatment:
Single exposure.
Post exposure period:
16, 24, 48 hours after treatment application.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 1000, 5000 mg/kg
Basis:
actual ingested
Tolerability test 1
Remarks:
Doses / Concentrations:
4000 mg/kg
Basis:
actual ingested
Tolerability test 2
Remarks:
Doses / Concentrations:
4000 mg/kg
Basis:
actual ingested
Mutagenicity test
No. of animals per sex per dose:
- In the tolerability test: 2/sex/group.
- In the mutagenicity test: 24/sex/group, except 8/sex/group in positive control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide.
- Route of administration: Oral (not specified).
- Doses / concentrations: 64 mg/kg cyclophosphamide in 20 mL CMC 0.5%.

Examinations

Tissues and cell types examined:
Bone marrow (erythrocytes) is harvested from the shafts of both femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the suitable dosage of the test substance to be applied in the mutagenicity assay.
Three groups of four Chinese hamsters (two females and two males) are treated with three different doses, one receiving the maximum dose of 5000 mg/kg, or the highest applicable dose, and the other two doses of 1/5 (1000 mg/kg) and 1/25 (200 mg/kg) of that amount respectively. The animals are treated with a single dose. The observation period corresponds to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. If all animals in all dose groups die in the first step, a second test is performed in which the highest dose given is 3/4 of the lowest used in the preceding test. If some of the animals in one of the dose groups die, the test is continued with a high dose corresponding to a predetermined fraction of that dose. Depending on the outcome the highest dose causing no death is used as the highest in the mutagenicity test, or if necessary the test is repeated with lower doses.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The preparation was administered orally to animals. Treatment consisted of a single application. 16, 24 and 48 hours after application 8 female and 8 male animals per sampling time were sacrificed by dislocation of the cervical vertebrae.

DETAILS OF SLIDE PREPARATION:
Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Grünwald solution for 3 minutes then in May-Grünwald solution/water 1/1 for 2 minutes. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 minutes. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS:
Prior to analysis the slides are coded. Thereafter the quality of staining is evaluated. The slides of five animals from each sex showing the best differentiation between mature and polychromatic erythrocytes are selected for later scoring. The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post treatment are examined. From the animals of the positive control group which are sacrificed 24 hours after application, the slides of five female and five male animals are scored. 1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei. To determine the mitotic activity of the red compartment, the ratio of polychromatic to normochromatic erythrocytes is calculated for each animal by counting a total of 1000 erythrocytes. A low proportion of polychromatic erythrocytes is indicative for a mitosis inhibiting activity of the test substance.
Evaluation criteria:
A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any sampling time.
Assay acceptance criteria:
- The quality of the slides must allow a clear differentiation between polychromatic and normochromatic erythrocytes.
- The result obtained with the positive control has to fulfil the criteria given for a positive response.
Statistics:
The significance of difference is assessed by Chi square-test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (Tolerability test)
- Dose range:
a) Test 1: 200, 1000, 5000 mg/kg
b) Test 2: 4000 mg/kg
- Clinical signs of toxicity in test animals: In Test 1 of the tolerability test the maximum dose (5000 mg/kg) caused the death of one animal. In Test 2 of the tolerability test the dose of 4000 mg/kg caused no death in a group of four animals.
- Rationale for exposure: Based on tolerability test.

RESULTS OF DEFINATIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): Refer to Table 1.
- Appropriateness of dose levels and route: One female animal of the 24 hours dosage group died during the treatment period.
- Statistical evaluation: There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 4000 mg/kg as compared with the negative control animals at all three sampling times.

Any other information on results incl. tables

Table 1. Micronucleus Test on Chinese Hamster Bone Marrow Cells*

Sacrifice

Treatment

Sex

PCE

NCE

Ratio of PCE/NCE

Number of PCE with micronuclei

% of PCE with micronuclei

16 h

Vehicle Control

F

559

441

1.3

0.6

0.06

M

564

436

1.3

0.6

0.06

test item

F

515

485

1.1

1

0.1

M

507

493

1

0.2

0.02

24 h

Vehicle Control

F

526

474

1.1

0.2

0.02

M

523

477

1.1

0.6

0.06

test item

F

505

495

1

0.2

0.02

M

501

499

1

0.8

0.08

48 h

Vehicle Control

F

529

471

1.1

0.4

0.04

M

501

499

1

0.8

0.08

test item

F

491

509

1

0

0

M

527

473

1.1

0.2

0.02

24 h

Vehicle Control

F

526

474

1.1

0.2

0.02

M

523

477

1.1

0.6

0.06

Positive control**

F

455

545

0.8

22

2.2

M

487

513

0.9

22.6

2.26

% = percentage; F = females; h = hours; M = males; NCE = normochromatic erythrocytes; PCE = polychromatic erythrocytes.

* test item administered at 4,000 mg/kg.

** positive control = cyclophosphamide.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated withthe test substance.