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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
June 8, 2000
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-mercaptoethanol
EC Number:
200-464-6
EC Name:
2-mercaptoethanol
Cas Number:
60-24-2
Molecular formula:
C2H6OS
IUPAC Name:
2-sulfanylethanol
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: ATOFINA
- Lot/batch No. of test material: 26/10/01
- Appearance: Colorless light yellow liquid
- Expiration date of the lot/batch: November 2002
- Purity: 99.852%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature and protected from light.

Test animals

Species:
mouse
Strain:
other: Swiss Ico. OF1 (IOPS Caw)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, I'Arbresle, France.
- Age at study initiation: approximately 6 weeks
- Housing: The animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, 94142 Alfortville, France). No contaminants were found in the sawdust.
- Diet: A04 C pelleted maintenance diet (UAR, 91360 Villemoisson-sur-Orge, France) was provided ad libitum. No contaminants were found in the diet.
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. No contaminants were found in the water.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70%
- Air changes: at least 12 cycles/hour of filtered non-recycled fresh air.
- Photoperiod: 12/12 (0:700 - 19:00)

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 / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
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 CO2 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