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: GLP and guideline study

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)
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
purity: 99.86%

Test animals

Species:
mouse
Strain:
other: Swiss Ico. OF1 (IOPS Caw)
Sex:
male/female
Details on test animals and environmental conditions:
Age of animals: on the day of treatment, the animals were approximately 6 weeks old.
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination to ensure that they were in good clinical conditions.
Acclimation: at least 5 days before the day of treatment.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex.
Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.
Upon their arrival at CIT, the animals were housed in an animal room, with the following enviromnental conditions: temperature: 22 +/- 2°C, relative humidity: 30 to 70%; light/dark cycle: 12 h/ 12 h (07:00 - 19 :00); ventilation: at least 12 cycles/hour of filtered non-recycled fresh air
The temperature and relative humidity were under continuous control and recording. The housing conditions (temperature, relative humidity and ventilation) and corresponding instrumentation and equipment were verified and calibrated at regular intervals. The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust. Sawdust is analyzed by the supplier for composition and contaminant levels.
All animals had free access to A04 C pelleted maintenance diet. Each batch of food is analysed by the supplier for composition and contaminant levels. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines).
No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to interfere with or prejudice the outcome of the study.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
isotonic solution
Details on exposure:
The test item was dissolved in the vehicle in order to achieve the concentrations of 5, 10 or 20 mg/mL and then homogenized using a magnetic stirrer .
The preparations were made immediately before use.
Duration of treatment / exposure:
- Preliminary toxicity test: injected twice
- Main study: test groups received 2 i.p. injections
Frequency of treatment:
two administrations
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 100, 200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
- Preliminary toxicity test: 3 males and 3 females
- Main study: 5 male and 5 female mice per group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Preparation of the bone marrow smears: At the time of sacrifice, all the animals were killed by C02 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).
Evaluation criteria:
For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.
Statistics:
When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to determine the X2 value (Lovell et al., 1989). When there was significant within group hete'rogeneity, then that group was compared with the control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969). The student "t" test was used for the PE/NE ratio comparison. Probability values of p<0.05 was considered as significant.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs, decreased PCE:NCE ratio
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test: no clinical signs at 100 mg/kg bw; at 200 mg/kg bw hypoactivity, dyspnea and/or piloerection noted in males and females, no mortality; at 500 mg/kg bw all mice died within 1 h after the first treatment.
Clinical signs in the main study: no clinical signs observed in the low and mid dose group 2 and 24 h after the 1st or the 2nd injection; hypoactivity was seen in the high dose group 2 h after the 1st and the 2nd application, no clinical signs 24 h after each injection.
Cytogenetic: no significant differences in MPE values between vehicle controls and treated females of all dose groups; no significant effects in males of the low and mid dose group; males of the high dose group showed a slight but statistically significant (p<0.05) increase in the frequency of MPE; but data generated in the additional analysis (further 2000 PE per animal evaluated) showed that there was no significant difference between males of the high dose group and the corresponding control group; combination of these data to the data previously generated (increased size of the samples analysed for high dose males and control males, 4000 PE) showed also no significant difference.
For males of the mid and the high dose group the PN/NE ratio decreased significantly indicating toxic effects of the test substance on bone marrow cells.

Any other information on results incl. tables

Cytogenetic summary table:

 Dose (mg/kg bw)  MPE/1000PE (standard deviation)  PE/NE ratio (standard deviation)
 vehicle (male)  0.2 (0.3)  0.7 (0.1)
 50 (male)  1.1 (1.9)  0.5 (0.2)
 100 (male)  0.8 (0.8)  0.5 (0.1) - p<0.05
 200 (male)  1.2 (0.6) - p<0.05  0.4 (0.2) - p<0.05
 positive control (male)  15.7 (4.8) - p<0.001  0.6 (0.2)
 vehicle (female)  0.5 (0.9)  0.7 (0.2)
 50 (female)  0.6 (0.7)  1.0 (0.2)
 100 (female)  0.9 (1.0)  0.9 (0.1)
 200 (female)  1.1 (1.1)  0.8 (0.2)
 positive control (female)  17.8 (6.3) - p<0.001  1.0 (0.2)

Results of additional analysis in males:

 Dose (mg/kg bw)  MPE/1000PE (standard deviation)  PE/NE ratio (standard deviation)
 vehicle  1.5 (1.1)  0.7 (0.1)
 200  1.1 (1.0)  0.4 (0.2) - p<0.05

Results of pooled data in males:

 Dose (mg/kg bw)  MPE/1000 PE (standard deviation)
 vehicle  0.9 (0.6)
 200  1.2 (0.7)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under our experimental conditions, the test item 2-MERCAPTOETHANOL does not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, with a 24-hour interval, at the dose-levels of 50, 100 or 200 mg/kg/day.