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
Study period:
1999-12-03 to 2000-01-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
2-tert.-butylphenol
IUPAC Name:
2-tert.-butylphenol
Constituent 2
Chemical structure
Reference substance name:
2-tert-butylphenol
EC Number:
201-807-2
EC Name:
2-tert-butylphenol
Cas Number:
88-18-6
Molecular formula:
C10H14O
IUPAC Name:
2-tert-butylphenol
Constituent 3
Reference substance name:
o.-tert.-butylphenol
IUPAC Name:
o.-tert.-butylphenol
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 2-tert.-butylphenol
- Physical state: liquid
- Analytical purity: 99.97%
- Purity test date: 2014-05-07
- Lot/batch No.: 1419
- Expiration date of the lot/batch: 05/2015
- Stability under test conditions: stable
- Storage condition of test material: under N2 in tightly closed container at a cool, well ventilated place
- Colour: clear

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: HARLAN NOSSAN S.r.l., Carezzana (MI), Italy
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation: 20.9 - 29.2 g
- Assigned to test groups randomly: animals were allocated to test groups, no further details mentioned
- Fasting period before study: overnight
- Housing: 5 animals/cage, by sexes, in clear polycarbonate cages measuring 35.5 x 23.5 x 19 cm with a stainless steel mesh lid and floor (Type 2b: Techniplast)
- Diet: Altromin MT diet ad libitum
- Water: drinking water ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1999-12-09 To: 1999-12-16

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: not stated
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
- Lot/batch no. (if required): batch no. 107H1649 obtained from Sigma Chemical Co.
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
no details mentioned


Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single treatment by oral gavage
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
250, 500, 1000 mg/kg body weight
Basis:
no data
No. of animals per sex per dose:
preliminary toxicity test: 2
main assay: 5 for the low and mid dose group and the positive control (24 h sampling time); 10 for the vehicle control and the high dose group (24 and 48 h sampling times)
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): not stated
- Route of administration: by gavage
- Doses / concentrations: solution of Mitomycin C (MMC, batch no. 088/AFA Kyowa Hakko Kogyo Co. Ltd., Tokyo) in sterile distilled water, concentration 3.0 mg/kg body weight

Examinations

Tissues and cell types examined:
Preparation of the bone marrow were made on slides.
Poly- and normochromatic erythrocytes and micronucleated cells (poly- and normochromatic) were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A dose-level equivalent to 80 % of the LD50 value was used as an estimate of the maximum tolerated dose-level.
- Criteria for selection of M.T.D.: Ratio of immature erythrocytes (PCE) to total erythrocytes (PCE+NCE), clinical signs and surviving of dosed animals. The M.T.D. should induce some tocix signs, such as the inhibition of bodyweight gain.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- Sampling times and number of samples: 24 h sampling time: five animals per sex from each group; 48 h sampling time: 5 animals per sex of the negative control and high dose group

DETAILS OF SLIDE PREPARATION:
- Bone marrow preparation: Animals of all groups were sacrified at appropriate sampling times. The femurs of animals were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Theslides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.


METHOD OF ANALYSIS:
- Slide evaluation: The slides were examined under low power (x 16 objective), one slide from each animal was selected according to staining and quality of smears. The slides were coded and where no depression of polychromatic erythrocytes was observed at least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.


OTHER:
Daily clinical observations of the treated animals were made for the justification for dose selection.
Evaluation criteria:
The test item is considered to induce micronuclei if a statistically significant increase in the micronuleus incindence in polychromatic erythrocytes (at P < 0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.
Where increases in the incidence of micronuleated PCE`s were observed which were statistically significant, but fall within the range of negative control values within the testing laboratory, then concurrent and historical control data were used to demonstrate that these increases did not have biological significance. Historical controls were included in the test report.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleated frequencies per 2000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at the highest dose level (1000 mg/kg bw)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Toxicity test 1: 1000 and 2000 mg/kg body weight (male/female)
Toxicity test 2: 1300 (female only), 1600 (male/female) and 2000 (male only) mg/kg body weight
- Clinical signs of toxicity in test animals:
Toxicity test 1:
2000 mg/kg b.w. showed moribund animals, ataxia, hunched posture, reduced activity, pale appearance, cold to touch, swollen abdomen, increased activity, tremors, piloerection for both sexes.
1000 mg/kg b.w. showed ataxia, hunched posture, reduced activity, pale appearance, swollen abdomen, piloerection for both sexes.
female mice of the treatment with 2000 mg/kg bodyweight died, the surviving male mice were observed for 48 hours and sacrificed.
Toxicity test 2:
All animals of both sexes and all dose levels showed the following clinical signs: moribund animals, convulsions, hunched posture, reduced activity, pale appearance and piloerection.
Two male mice, treated with 2000 mg/kg b.w., one male mouse, treated with 1600 mg/kg b.w. and one female mouse, treated with 1300 mg/kg b.w. died. Animals were observed for 24 hours and sacrificed.
- Evidence of cytotoxicity in tissue analyzed: The ratio of immature erythrocytes (PCE) to total erythrocytes (PCE+NCE) showed mild depression bone marrow erythopoietic cell division following treatment with the test item, both in male and female animals.
- Rationale for exposure: Based on the results of the range finding study dose-levels of 250, 500 and 1000 mg/kg body weight were selected for the definitive study.



RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No marked increases in the numbers of micronucleated PCE's were observed in any treatment group and any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system.
- Ratio of PCE/NCE: A moderate inhibitory effect to erythropoietic cell division was observed at the high dose-level in male animals, at 24 and 48 hours sampling time, and in female animals at 48 hour sampling time only.
- Appropriateness of dose levels and route: Clinical signs, mortality and bone marrow cell toxicity provide evidence of bioavailibitity of the test substance and adequate exposure.
Clinical signs: 250 mg/kg b.w.: hunched posture and piloerection; 500 mg/kg b.w.: tremors, difficulty in movements and piloerection; 1000 mg/kg b.w.: hunched posture, difficulty in movements, tremors and piloerection. A number of male and female animals were found moribund the day of treatment, the surviving animals appeared to make full recovery before the final sampling time.
Mortality: Three female animals of the highest dose level (1000 mg/kg b.w.) died prior to the 24 hours sampling time, 2 of them were substituted by reserve animals.
- Statistical evaluation: No statistically significant increase in the incidence of micronucleated PCE¿s over the control value was observed at any sampling time for male and female animals combined. A statistically significant heterogeneity was observed within male animals from the high treatment group at 48 hour sampling time.

Any other information on results incl. tables

Table: Summary of incidences of micronucleated cells, both sexes

 dose-level [mg/kg]  PCE/NCE ratio     incidence of micronucleated cells / 1000 cells         
      polychromatic      normochromatic      
    24 h  48 h  24 h  48 h  24 h  48 h
vehicle control   0.821 0.985  1.5 ± 0.1   1.4 ± 0.2  0.5 ± 0.2  0.6 ± 0.2
 250  0.745    1.2 ± 0.2    0.7 ± 0.1  
 500  0.767    0.9 ± 0.2    0.5 ± 0.1  
 1000  0.636  0.563  1.1 ± 0.2  1.4 ± 0.4  0.6 ± 0.1  1.0 ± 0.2
 positive control  0.749    10.4 ± 1.6    0.7 ± 0.2  

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The study has been conducted with a structurally closely related material, the only difference being that the alkyl chain is in the ortho-position rather than the p-position. Following treatment with o-tert-Butylphenol, no statistically significant increase in the incidence of micronucleated PCE¿s over the control value was observed at any dose-level, at any sampling time.
Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were observed, indicating the correct functioning of the test system.
It was concluded that, under the reported experimental conditions, o-tert-Butylphenol administered orally at the selected dose-levels to male and female mice, did not induce micronuclei in the polychromatic erythrocytes. Clinical signs and mortality observed at the highest dose-level, provide evidence of bioavailability of the test item and adequate exposure.