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:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
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.
Qualifier:
according to
Guideline:
other: EPA Health Effects Testing Guidelines 50 FR 3939 and Revision of TSCA Test Guidelines 52 FR 19080, 52 FR 26150, 52 FR 34654
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect at the time of study conduct.
Qualifier:
according to
Guideline:
other: The inhalation portion of the study was conducted according to EPA Health Effects Testing Guideline 50 FR 39397 and Revision of TSCA Test Guidelines 54 FR 21064
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: 99.68% minimum

Test animals

Species:
rat
Strain:
other: CD®BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 48 days old
- Weight at study initiation: Males: 242.7-293.9 g (mean 271.2 g); Females: 188.4-231.2 g (mean 201.4 g)
- Fasting period before study: No
- Housing: Individually in standard wire mesh cages, except during exposure
- Diet (e.g. ad libitum): Purina Certified Rodent Chow® #5002 ad libitum, except during exposure
- Water (e.g. ad libitum): ad libitum, except during exposure
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Humidity (%): 50±10%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

Administration / exposure

Route of administration:
inhalation: vapour
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Three chambers of approximately 750 L and one chamber of approximately 1000 L
- Method of holding animals in test chamber: whole-body; no further details reported
- Source and rate of air: Conditioned house air
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: The chambers were operated in a one-pass, flow-through mode
- Temperature, humidity, pressure in air chamber: Temperature was 20-24°C, relative humidity was 31-70%, pressure was not reported, and mean oxygen concentration was 20.6-21.0 during exposure
- Air flow rate: Mean airflow rate was 207-208, 114-119, 115, 152-155 L/min for the 0, 2000, 3500, and 7000 ppm exposures, respectively
- Air change rate: Not reported
- Method of particle size determination: Not reported
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: Test vapour was generated by metering the test material into a J-tube filled with glass beads. Heated air (approximately 60°C) was blown through the glass beads to evaporate the liquid test material. The resulting vapour was diluted with filtered conditioned air before entering the test chamber. Conditioned heated air was metered into the control chamber in a similar manner. Chamber atmospheres were analyzed by gas chromatography at 30-minute intervals during each 6-hour exposure period. Gas samples were analyzed with a gas chromatograph equipped with a flame ionization detector. The temperature of the injection port was ambient, and the temperature of the flame ionization detector was 250°C. Nitrogen was utilized as the carrier gas. Samples were chromatographed isothermally at 135°C on a stainless steel column packed with Fluorocol 60/80 Carbopak B. The atmospheric concentration of the test substance was determined by comparing the detector response of the chamber samples to that of gas standards through the use of standard curves.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
2 consecutive days
Frequency of treatment:
6 hours/day
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2000, 3500, 7000 ppm (0; 20618; 36081; 72162 mg/m3, respectively)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1987 (Day 1) and 2008 (Day 2) ppm (20485 and 20704 mg/m3, respectively)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
3292 (Day 1) and 3575 (Day 2) ppm (33946 and 36857 mg/m3, respectively)
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
7125 (Day 1) and 6984 (Day 2) ppm (73461 and 72002 mg/m3, respectively)
Basis:
analytical conc.
No. of animals per sex per dose:
12/sex/concentration
Control animals:
yes
Positive control(s):
- Positive Control: Cyclophosphamide
- Justification for choice of positive control(s): Not reported
- Route of administration: ip injection
- Doses / concentrations: 25 mg/kg (5 mL/kg dosing volume)

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCEs) and Normochromatic erythrocytes (NCEs) from rat bone marrow taken from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The selection of the maximum exposure level was based on available toxicity data and a preliminary rangefinding experiment. In rats, the ALD for a single 4-hour exposure was 10000 ppm. In a 2-week study, the NOAEL for repeated exposure was 4000 ppm (the highest concentration evaluated). The toxicity of repeated exposures greater than 4000 ppm was determined as part of a rangefinding experiment for a subchronic study. In one experiment, 5 male and 5 female rats per group were exposed to atmospheric concentrations of up to 7000 ppm for approximately 4 to 6 hours per day on 3 or 4 consecutive days. As chamber concentrations increased above 3500 ppm on each day, seizure-like clinical signs were observed. At the end of the third exposure, one animal at 7000 ppm was found dead. Upon review of this data, the maximum concentration that appeared acceptable for use in the micronucleus assay was 7000 ppm. A rangefinding experiment was then conducted for the micronucleus study to ensure that animals given 2 exposures at 7000 ppm would survive until a 48-hour post-exposure sacrifice. Five male and 5 female rats were exposed to 7000 ppm for 6 hours per day on 2 consecutive days. On both days, 50-75% of the animals were observed to be convulsing within 1-1.5 hours of the initiation of exposure, however, all animals survived until the end of the rangefinding experiment. Based on this information, the maximum concentration selected for the micronucleus assay was 7000 ppm. Additional lower treatment concentrations of 3500 and 2000 ppm were also selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Groups of 5 or 6 males and females form the negative control and each treatment group were sacrificed approximately 24 and 48 hours after the final exposure. The positive indicator group, consisting of 5 male and 5 female rats were concurrently treated on 2 consecutive days and were sacrificed approximately 24 hours after the second exposure.

DETAILS OF SLIDE PREPARATION: Immediately following sacrifice, the marrow from 1 femur of each animal was aspirated and flushed into prewarmed (37°C) foetal bovine serum. The marrow was collected by centrifugation at approximately 200 x g for 5 minutes. Most of the supernatant was removed and the cells were resuspended in the remaining serum. An automatic blood smearing instrument was used to make the bone marrow smears. At least 2 slides per animal were prepared and fixed in absolute methanol for 8 minutes. The slides were stained for 3 minutes in acridine orange. Prior to scoring, a coverslip was floated on each slide using phosphate buffer.

METHOD OF ANALYSIS: Representative slides from each animal were examined in a blind manner using incident light fluorescence microscopy. Only cells showing good morphology and staining were selected for scoring. PCEs were identified by their characteristic reddish staining; NCEs appeared dark green. Two thousand PCEs per animal were scored for the presence of micronuclei. Cellular inclusions that were irregularly shaped or stained, or were not in the focal plane of the cell were considered artefacts. The unit of scoring was the micronucleated cell. PCEs containing more than one micronucleus were counted as a single micronucleated cell. The number of micronucleated NCEs seen in the optic fields scored to obtain one thousand PCEs was also recorded. The proportion of PCEs among one thousand erythrocytes was determined for each animal.
Evaluation criteria:
The test substance was classified as clastogenic (positive) if both of the following effects were reproducible (i.e., evident in 2 or more trials under activated or nonactivated conditions: (1) the test substance produced a statistically significant increase in percent of abnormal cells as compared to the negative (solvent) control at one or more test concentrations (p≤0.05); and (2) there was a statistically significant dose-related increase in percent abnormal cells (p≤0.01). The test substance was classified as nonclastogenic (negative) if the following criteria were met: (1) the test substance did not produce a statistically significant increase in percent abnormal cells at any concentration tested; and (2) there was no statistically significant dose-related increase in percent abnormal cells. Results not meeting those criteria for positive or negative assessments were evaluated on a case-by-case basis.
Statistics:
Data for the proportion of PCEs among 1000 erythrocytes (PCE frequency) and the proportion of micronucleated PCEs (MNPCEs) among 2000 PCEs (MNPCE frequency) for all dose levels and sacrifice intervals were transformed prior to statistical analysis using the arcsin square root transformation. Transformed data for each variable (PCE or MNPCE frequency) were analyzed separately for normality of distribution using the Shapiro-Wilkes test. The arcsin square root transformations for PCE and MNPCE frequency were found to be normally distributed. Therefore, these data were analyzed using one way ANOVA. The VMS program “Dunnettstats” was used for all ANOVA calculations and for making individual comparisons to the control. Positive indicator data were not included in the evaluation of normality of distribution. Weight gain data were assumed to be normally distributed and were analyzed using Analysis of Variance (ANOVA). Data from each sex and sacrifice time were analyzed separately, and individual comparisons to the control were made using each animal as the experimental unit. All analyses were conducted at a significance level of 5%. Positive indicator data were analyzed separately.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Clinical signs were observed.
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
-Moderate to severe signs of toxicity were observed during exposures on days 1 and/or 2 in the 3500 and 7000 ppm treated groups. On day 1, clinical signs in both groups included convulsions, prostrate posture, and lethargic behaviour. The convulsions occurred throughout the exposure, but were more frequent during the first hour. In addition, the 7000 ppm treated animals also exhibited barrel rolling, piloerection, and salivation. Day 1 post-exposure signs for the 7000 ppm treated group included hyperactivity and stained fur. No post-exposure signs were evident in the 3500 ppm treated group and no clinical signs of toxicity were observed in the 2000 ppm group on day 1. On day 2, clinical signs for the 7000 ppm treated group were similar to those observed on day 1, however, for the 2000 and 3500 ppm groups, lethargy was the only sign observed during the exposure. Day 2 post-exposure signs for the 7000 ppm treated group included stained and/or ruffled fur. No post-exposure signs of toxicity were observed in the 2000 or 3500 ppm treated groups on day 2.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability). Under the conditions of this assay, the test material did not induce micronuclei in bone marrow cells of rats.
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 and female CD®BR rats were placed in exposure chambers and exposed whole-body to target concentrations of 0, 2000, 3500, and 7000 ppm for 6 hours per day on 2 consecutive days. Bone marrow smears were prepared approximately 24 and 48 hours after the second exposure and 2000 polychromatic erythrocytes per animal were evaluated for the presence of micronuclei. No statistically significant increases in the number of micronucleated polychromatic erythrocytes were observed at any concentration tested. In addition, no significant decreases in the ratio of young polychromatic erythrocytes to mature normochromatic erythrocytes were observed. Under the conditions of this study, the test material is negative.