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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.54%

Test animals

Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 287 - 383 g
- Housing: housed on mobile rat cage racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): at least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light followed by 12 hours darkness

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
dried filtered air
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2 tiered nose-only exposure chamber
- Source and rate of air: dry filtered air
- System of generating vapour: atmospheres were generated by metering appropriate amounts of liquid test substance on to the top of a water bath heated condenser column (45°C) using a Hamilton Microlab pump. A counterflow of clean dry compressed air (25 L/min) was used to pass the resultant vapour to a 2 tiered nose-only exposure chamber

TEST ATMOSPHERE
- Brief description of analytical method used: Test atmospheres were sampled by trapping the test substance on Carbotrap C 20/40 mesh in a desorption tube, followed by thermal desorption and quantification using gas chromatography.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
1 day
Frequency of treatment:
4 hours/day
Post exposure period:
24 hours for dose levels 200, 400, and 800 ppm
48 hours for dose level 800 ppm
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200, 400, and 800 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
224, 387, and 775 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
200 and 400 ppm - 5/dose
800 ppm - 10/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- positive control: cyclophosphamide

- Route of administration: The positive control substance was prepared as a solution in double deionised water and was dosed at a volume of l0 mL/kg bodyweight. A group of rats was given a single oral dose of the positive control substance to coincide with the end of the inhalation exposure period.
- Doses / concentrations: l0 mL/kg bodyweight

Examinations

Tissues and cell types examined:
immature erythrocytes from rat bone marrow taken from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The highest concentration represents the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities observed in a previous rat toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Groups of male rats were exposed by the inhalation route for a single 4 hour period to either dried filtered air or the test substance at nominal concentrations of 200, 400 and 800 ppm. Bone marrow samples were taken approximately 24 hours after the end of the exposure period for the vehicle control and the test substance at dose levels of 800, 400 and 200 ppm and 48 hours after the end of the exposure period for the vehicle control and the test substance at 800ppm. Bone marrow samples were taken 24 after dosing for the positive control.

DETAILS OF SLIDE PREPARATION: Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide. The slides were allowed to air dry and were stained with acridine orange.

METHOD OF ANALYSIS: Slides were coded and scored blind. Two thousand immature erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of immature to mature erythrocytes in a sample of 1000 erythrocytes.

Evaluation criteria:
The data have been interpreted as follows:
a) No statistically significant increase in the incidence of micronucleated immature erythrocytes above concurrent vehicle control incidences -NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated immature erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range - NEGATIVE.
c) A statistically and biological significant increase in the incidence of micronucleated immature erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated immature erythrocytes which is statistically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.
Statistics:
The data for the incidence of micronucleated immature erythrocytes were transformed using a square root transformation, prior to analysis. The data for the percentages of immature erythrocytes were transformed using the double arcsine transformation of Freeman and Tukey, prior to analysis.
Analyses were carried out using the MIXED procedure in SAS. Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Clinical signs: stains around nose, decreased activity, tiptoe gait, abnormal breathing, reduced stability and reduced response to sound

Mortality: one male found dead before sampling time, macroscopic abnormalities included mesenteric/renal lymph nodes, mottled/pale areas on liver, pelvic dilatation of the kidney, distended bladder, gas-filled caecum and a diaphragmatic hernia on the liver

Genotoxicity: No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes were observed. No statistically significant differences in the percentage of immature erythrocytes between vehicle control and test substance were observed

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Under the conditions of the test, the test substance is neither clastogenic nor aneugenic in the rat bone marrow micronucleus test.
Executive summary:

An inhalation micronucleus study was conducted in rats to determine whether the test material induces an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow. In this study, groups of male rats were placed in exposure chambers and exposed nose-only to target concentrations of 200, 400, and 800 ppm for 4 hours per day on one day. Bone marrow smears were prepared approximately 24 and 48 hours after exposure and 2000 immature erythrocytes per animal were evaluated for the presence of micronuclei. 

No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes were observed. No statistically significant differences in the percentage of immature erythrocytes between vehicle control and test substance were observed. Under the conditions of the test, the test substance is neither clastogenic nor aneugenic in the rat bone marrow micronucleus test.