Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-06-13 to 1989-08-15
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
other: mouse micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
α,α,α-trifluoro-4-nitro-m-cresol
EC Number:
201-818-2
EC Name:
α,α,α-trifluoro-4-nitro-m-cresol
Cas Number:
88-30-2
Molecular formula:
C7H4F3NO3
IUPAC Name:
4-nitro-3-(trifluoromethyl)phenol
Test material form:
solid

Test animals

Species:
mouse
Strain:
ICR
Details on species / strain selection:
This healthy, random bred strain was selected to maximize genetic heterogeneity and at the same time assure access to a common source.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD., USA
- Age at study initiation: adult, ~8.5 weeks
- Weight at study initiation: ♂: 29.0 - 38.5 g; ♀: 21.8 - 28.3 g
- Assigned to test groups randomly: yes

- Housing: group-housed up to five per cage in sanitary polycarbonate cages.
- Diet: ad libitum, Purina Certified Laboratory Chow #5002
- Water: ad libitum
- Acclimation period: at least 7 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: solubility of the test item
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions for the assay were prepared by making a 80 mg/mL stock for the high dose (800 mg/kg). Dilutions of this stock were prepared for the 400 and 80 mg/kg dose levels.
Frequency of treatment:
single exposure
Post exposure period:
treatment group: 24, 48, and 72 h
positive and vehicle control group: 24 h
Doses / concentrationsopen allclose all
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
800 mg/kg bw/day
No. of animals per sex per dose:
5
Remark: A second group of animals (designated Secondary Dose Group, 10 animals/sex/dose) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral gavage
- Dose: 80 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The dose levels used in this assay were based upon the results of a previously conducted dose range finding assay.

TREATMENT AND SAMPLING TIMES: The doses selected for this assay were 80, 400, and 800 mg/kg body weight and were administered by a single oral gavage.

DETAILS OF SLIDE PREPARATION: At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both tibiae were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 3 mL fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on slides and air-dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water (Schmid, 1975). After being air-dried, the slides were coverslipped using Depex® mounting medium.

METHOD OF ANALYSIS: The coded slides were scored for micronuclei and the polychromatic (PCE) to normochromatic (NCE) cell ratio. Standard forms were used to record these data. Where possible, one thousand PCEs were scored from each animal. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCEs present in the scored optic field. The normal frequency of micronuclei in this mouse strain is about 0.0 - 0.42. The frequency of PCEs versus NCEs was determined by scoring the number of NCEs observed in the optic fields while scoring the 1000 PCEs for micronuclei.
Evaluation criteria:
The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei had sharp borders and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micro-nucleated PCE, not two (or more) micronuclei. The staining procedure permitted the differentiation by color of PCEs and NCEs (bluish-grey and red, respectively).
The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant positive response for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on scientific judgment.
Statistics:
Data were summarized to include tables indicating the individual animal results and in tables with animal results summarized by sex and dose groups at the different time points. The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion, Sokal and Rohlf, 1981). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different from the negative control. Analyses were performed separately for each harvest time and sex combination, and also at each harvest time for the sexes combined.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
mortality, within 24 h after administration, 11 animals died in the 800 mg/kg dose group and 1 animal in the 400 mg/kg dose group.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No
- Ratio of PCE/NCE: see: Any other information on results incl. tables
- Statistical evaluation: The analysis of the data was performed using an Analysis of Variance on the square root arcsine transformation which was performed on the proportion of cells with micronuclei per animal (square root arcsine proportion, Sokal and Rohlf, 1981). Once the Analysis of Variance had been performed, Tukey's Studentized range test (HSD) with adjustment for multiple comparisons was used at each harvest time to determine which dose groups, if any, were significantly different from the negative control.

Any other information on results incl. tables

 

Treatment

Dose

Harvest Time (h)

% Micronucleated PCEs
Mean of 1000/Animal ± S.E. (1)

Ratio PCE:NCE
Mean ± S.E.

Total

Neg. Control

10.0 mL/kg

24

0.12 ± 0.04

0.06 ± 0.02

0.09 ±0.02

0.36 ± 0.03

0.45 ± 0.04

Pos. Control

80 mg/kg

24

1.54 ± 0.68*

1.38 ± 0.41*

1.46 ± 0.38*

0.55 ± 0.01

0.36 ±0.04

Test Substance

80 mg/kg

24

0 06 ± 0.02

0.08 ± 0.05

0.07±0.03

0.34 ± 0.04

0.54 ± 0.05

48

0.06 ± 0.04

0.02 ± 0.02

0.04 ± 0.02

0.48 ± 0.05

0.56 ± 0.08

72

0.14 ± 0.07

0.04 ± 0.04

0.09 ± 0.04

0.35 ± 0.03

0.49 ± 0.06

400 mg/kg

24

0.02 ± 0.02

0.04 ± 0.02

0.03 ± 0.02

0.50 ± 0.10

0.37 ± 0.06

48

0.06 ± 0.04

0.08 ± 0.06

0.07 ± 0.03

0.46 ± 0.03

0.62 ± 0.09

72

0.00 ± 0.00

0.05 ± 0.04

0.03 ± 0.02

0.38 ± 0.02

0.35 ± 0.03

800 mg/kg

24

0.10 ± 0.03

0.02 ± 0.02

0.06 ± 0.02

0.52 ± 0.15

039 ± 0.05

48

0.01 ± 0.05

0.16 ± 0.05

0.13 ± 0.04

0.55 ± 0.04

0.47 ± 0.06

72

0.02 ± 0.02

0.10 ± 0.04

0.06 ± 0.03

0.43 ± 0.06

0.31 ± 0.03

(1) One male in the 24 h 800 mg/kg group had only 491 PCEs analyzed for micronuclei.

* Significanty greater than the corresponding negative control, p < 0.05.

Applicant's summary and conclusion

Conclusions:
In an in vivo mouse micronucleus test similar to EPA OPP 84 -2, the test substance showed no clastogenic potential.
Executive summary:

The objective of this in vivo assay was to evaluate the ability of the test substance to induce micronuclei in bone marrow polychromatic erythrocytes of mice (strain ICR). The study was conducted similar to the EPA OPP 84-2 guideline. The test article was suspended in corn oil and dosed by oral gavage at 80, 400, and 800 mg/kg based upon the results of a previously conducted dose range finding assay. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. The animals were dosed with the test article and were euthanatized 24, 48 and 72 hours after dosing for extraction of the bone marrow. A second group of animals (designated Secondary Dose Group, 10 animals/sex/dose) was also assigned to the study and was dosed with the high dose of the test article. These animals were only used in the assay as replacements for any which died in the primary dose group. Vehicle (corn oil) and positive control (cyclophosphamide, 80 mg/kg bw) groups euthanatized 24 hours after dosing were included in the assay.

All animals were observed immediately after dosing and periodically throughout the duration of the assay for toxic symptoms and/or mortalities. Within ten minutes of dosing nine in the 800 mg/kg dose groups and one male in the 400 mg/kg dose groups had expired. Approximately 1.5 hours after dosing one additional male in the high dose group was found dead. The following morning, approximately 19 hours after dosing, one in the 800 mg/kg dose group was found dead. All other animals appeared normal and remained healthy until the appropriate harvest times. Apparent test article induced bone marrow toxicity was observed in one male in the 24 hour high dose group, since only 491 PCE could be analyzed for micronuclei. The data from this animal were included in the statisitical analysis although no difference in the evaluation of the test article would occur by its exclusion. The criteria for the identification of micronuclei were those of Schmid (1976). No statistically significant reduction in the PCE:NCE ratio relative to the negative control was detected at any dose level. The positive control, CP, induced significant increases in micronucleated PCEs in both sexes, with means and standard errors of 1.54 ± 0.68 and 1.38 ± 0.41 for the males and females, respectively.

The test substance did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay and is considered negative in the mouse bone marrow micronucleus test.