Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

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other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Quality of Life, Utrechtseweg 48, 3711 AJ, Zeist, the Netherlands
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Storage conditions: ambient temperature (15-25°C)

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sultzfeld, Germany
- Age at study initiation: 8-10 weeks
- Weight at study initiation: Mean weight: Negative control: 31.55g; Treated: 31.45g; Postive control: 31.20g
- Fasting period before study: none
- Housing: Sterilized macrolon cages with bedding of wood shavings (Lignocel, type 3/4, Rettenmaier, Rosenberg, Germany) with strips of paper (Enviro-dri, Lillico, Betchworty, England)
- Diet: ad libitum Rat & Mouse No.3 Breeding Diet, RM3 (SDS Special Diets Services, Witham, England)
- Water: ad libitum tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2 deg C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes/hr
- Photoperiod (hrs dark / hrs light): 12 and 12

Administration / exposure

Route of administration:
inhalation: gas
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation chamber consisted of a cylindrical stainless steel column, surrounded by a transparent cylinder with a volume of ca. 50 litres. The column consisted of 2 mixing chambers, a rodent tube section underneath, and the exhaust section at the bottom. The rodent tube section had 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, and measurement of oxygen, temperature and relative humidity. Only the nose of the rats protruded into the interior of the column.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders, positioned radially through the outer hood around the central column. The remaining ports were closed.
- System of generating test atmosphere: The inhalation equipment was designed to expose mice to a continuous supply of fresh test atmosphere. Liquid test material was evaporated in a temperature-controlled brass tube. Compressed dry nitrogen was used to pressurize the cylinder containing the test material. Before entering the top of the inhalation chamber, the test substance was mixed with humidified compressed air. Oxygen was added to ensure an oxygen concentration of at least 19.5%. The animals were placed in the exposure unit after stabilization of the test atmosphere concentration. The period between the start of the generation and the start of the exposure was about 103 minutes. The exposure unit for the control animals was supplied with a rotameter-controlled stream of dry compressed air only.
- Temperature, humidity, oxygen in air chamber: 21.3-23.1°C, 49.5-55.4% relative humidity, 20.6 - 20.7% oxygen
- Air flow rate: 15.6 L/min

ANALYSIS OF TEST ATMOSPHERE
The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser. The responses of the analysers were recorded on a PC every minute using a CAN transmitter. Test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser through a sample line. The mean response was calculated by averaging values read every minute. Prior to the actual study, the total carbon analyser was calibrated for the target concentration by sampling from 4 concentrations in a range including the target concentration. The concentrations were prepared by mixing mass flow controlled amounts of air, oxygen and test material.
Duration of treatment / exposure:
4 hrs
Frequency of treatment:
once
Post exposure period:
24 and 48 hrs
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
48719
Basis:
analytical conc.
No. of animals per sex per dose:
5 males/time point (negative control, test material), 5 positive control males
Control animals:
yes, sham-exposed
Positive control(s):
mitomycin C;
- Justification for choice of positive control(s): positive in this assay
- Route of administration: ip injection
- Doses / concentrations: 0.75 mg/kg-bw

Examinations

Tissues and cell types examined:
bone marrow smears, 4 smears per animal were prepared, at least one slide per animal was examined
Details of tissue and slide preparation:
- Sacrifice, bone marrow sampling and processing: At 24 hours after the end of the exposure, 5 negative control, 5 test animals and 5 positive control animals were sacrificed by cervical dislocation. At 48 hours after the end of the exposure, 5 negative control and 5 test animals were sacrificed. Immediately following sacrifice, the bone marrow cells of both femurs were collected into foetal calf serum and processed into glass drawn smears. Four smears per animal were prepared air dried and fixed in methanol. All smears were stained with a May-Grunwald/Giemsa solution. One or more slides per animal were used for analysis.
- Microscopic examination of bone marrow smears: The slides were randomly coded by a person not involved in the scoring of slides. The slides were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines, taking care that areas selected for evaluation were evenly distributed over the whole smear. The following criteria were used for the scoring of cells:
Polychromatic erythrocyte (PE) is an immature erythrocyte that contains ribosomes. Normochromatic erythrocyte (NE) is a mature erythrocyter that lacks ribosomes. A micronucleus is a small, normally round nucleus with a diameter of approx. 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.
The numbers of PE and NE were recorded in a total of 200 erythrocytes (E) per animal. If micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 200 E (PE +NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number NE.
Evaluation criteria:
- The study was considered valid if positive controls showed a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls were within the historical range.
- A test substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus, if it showed a positive response, namely: the mean number of MPE/2000 PE was statistically significantly higher compared to the negative control and was at least 6 MPE/2000 PE.
- A test substance was considered to be negative if it produced no positive response.
- The test substance or its metabolites were considered to be cytotoxic to the bone marrow via general circulation, if the test substance statistically significantly reduced the mean number of PE.
- Both statistical significance and biological relevance were considered together in the evaluation.
Statistics:
Initially a one-way ANOVA was preformed. If it yielded a positive result (p<0.05) it was followed by pooled error variance t-tests or, if variences were not homogenious, separate variance t-tests. All statistical tests were preformed using SAS V9.1 statistical software.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Behaviour, clinical signs and mortality: Clear restless (graded as slight) was seen in all animals at every observation point during exposure. No abnormalities were observed in mice exposed to the control atmosphere. In a preliminary experiment, one male mouse was exposed for 4 hours to a target concentration of 50000 ppm. Besides slight restlessness and breathing at an increased rate (graded as slight), no signs of toxicity were observed and mortality did not occur.
- Statistical analysis of micronucleus results: At time point 24 and 48 hours after treatment there was no statistically significant difference in the mean number of MPE/2000E between the test substance group and the negative control group. The mean number of MPE/2000 PE in the negative control group was within the historical range. The mean number of MPE/2000E in the positive control was statistically significantly (p<0.0001) increased compared with the negative control group. The mean number of PE in the test substance group did not differ statistically significantly from that in the negative control group. The mean number of PE in the positive control group was statistically significantly lower (p = 0.0015) than that in the negative control group. This indicated that the positive control substance Mitomycin C induced cytotoxicity to the bone marrow.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It was concluded that inhalation exposures to the test substance at a concentration of 50000 ppm for 4 hours, resulting in a dose of about 65800 mg/kg body weight, which was far in excess of the limit dose of 2000 mg/kg bodyweight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow cells of male mice under the conditions of this study.
Executive summary:

In a GLP-compliant erythrocyte micronucleus test, performed according to OECD guideline 474, two groups of 10 male CD-1 mice were exposed to the test substance at concentrations of 0 or 50000 ppm via inhalation for 4 hours. 24 and 48 hours after exposure 5 mice per group were sacrificed. A positive control group consisted of 5 males treated with a single intraperitoneal dose of mitomycin C (0.75 mg/kg bw) and sacrificed 24 hours after injection. The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in the positive control group showed a statistically significant increase compared to the negative control group. The mean number of MPE per 2000 PE in the negative control group was within the historical control range. Therefore, the study was considered valid. Both at 24 and 48 hours after inhalation exposure, no statistically significant differences between the test substance and the negative control group were found with respect to the mean number of PE per 200 erythrocytes (E) and the mean number of MPE per 2000 PE. It was therefore concluded that, under the conditions of this study, inhalation exposure to the test substance for 4 hours at a concentration of 50000 ppm, resulting in a dose of about 65800 mg/kg body weight, which far exceeded the limit dose of 2000 mg/kg body weight, did not induce damage to the chromosomes and/or mitotic spindle apparatus (micronuclei) in the bone marrow target cells of male mice.