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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
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 compliant guideline study, adequate for evaluation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Staatstoezicht op de Volkgezondheid, 22 March 1993
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Aqueous solution containing ca. 70wt% TBHP
- Clear, colourless aqueous liquid

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Male and female young adult Swiss mice (Charles River CD-1 strain) were obtained from Charles River Wiga GmbH, Sulzfekld, Germany and housed in Makrolon cages with bedding of sterilized softwood chips. Males were housed individually and weight 28-35 g at the start of treatment, females were group housed (5 / cage) and weighed 22-28 g at the start of treatment. Fresh pelleted diet (TNO Rodent G diet) and tap water were available ad libitum.

Administration / exposure

Route of administration:
intravenous
Vehicle:
saline
Details on exposure:
The i.v. route (tail vein) was selected in order to maximise the bioavailability of the test substance.
Duration of treatment / exposure:
24, 48 and 72 hr
Frequency of treatment:
single i.v. treatment
Post exposure period:
24, 48 and 72 hr
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bwt
Basis:
nominal conc.
Dose levels selected on the basis of 2 preliminary i.v. range finding toxicity trials
Remarks:
Doses / Concentrations:
70 mg/kg bwt
Basis:
other: actual dose
No. of animals per sex per dose:
Test groups: 15 male, 15 female
Positive controls: 5 male
Control animals:
yes, concurrent vehicle
Positive control(s):
A concurrent positive control group (5 males) was given mitomycin C (1.5 mg/kg bwt) by i.p. injection and sacrificed 24 hours post-treatment.

Examinations

Tissues and cell types examined:
All animals were observed for clinical signs 1-4 hr post-treatment.
Details of tissue and slide preparation:
Animals (5/sex) were sacrificed by cervical dislocation at 24, 48 and 72 hr post-injection. Femoral bone marrow was immediately collected into foetal calf serum, and 2 air-dried, methanol fixed smears were prepared per animal and stained with May-Grunwald Giemsa.
Evaluation criteria:
The slides were randomly coded and scored blind for:
- the number of polychromatic (PE) and normochromatic erythrocytes (NE) present in a total of 1000 erythrocytes (E) per animal.
- the number of micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE) per 1000 erythrocytes
Statistics:
The number of incidence of MPE per 1000 PE and the number of PE per 1000 E from the vehicle control and test groups was analysed using 3-way ANOVA with sex (male, female), group (control, test) and time (24 hr, 48 hr, 72 hr) used as factors.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
(consistent with TK data indicating negligible systemic distribution)
Toxicity:
yes
Remarks:
seen in rangefinder at 200 mg/kg bwt
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The incidence of micronucleated polychromatic erythrocytes (MPE) per 1000 polychromatic erythrocytes (PE) did not differ significantly from that of the vehicle controls at any sacrifice time indicating no genotoxic effect on bone marrow cells. The incidence of MPE in the positive control group was increased approx. 22-fold.

The incidence of polychromatic erythrocytes (PE) per 1000 erythrocytes in male mice (PE) did not differ significantly from that of the vehicle controls at any sacrifice time indicating no cytotoxic effect of TBHP on bone marrow. In female mice at 48 hr and 72 hr the number of PE per 1000 E was increased slightly (+24% at 48 hr, +36% at 72 hr; P0.05) suggesting a weak interaction with bone marrow.

These findings are consistent with toxicokinetic data that indicate that TBHP is rapidly converted to tert-butanol in vivo, limited any potential for systemic genotoxic effects.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
TBHP was not genotoxic in the mouse micronucleus test after i.v. injection.
Executive summary:

The clastogenic potential of TBHP toward bone marrow cells was investigated in male and female mice following i.v. injection (nominal dose 100 mg /kg bwt; actual dose 70 mg TBHP /kg bwt). Femoral bone marrow was collected 24, 48 and 72 hr post-injection, and preparations examined blind for the presence of normal or micronucleated polychromatic and normochromatic erythrocytes. The study was GLP-compliant and followed EU Method B.12 (guideline study). The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes did not differ significantly from that of the vehicle controls at any timepoint indicating no genotoxic effect on bone marrow cells. The incidence of micronucleated polychromatic erythrocytes in a positive control group (cyclophosphamide) was increased approx. 22-fold. There was no change in the incidence of polychromatic erythrocytes in TBHP-treated male mice at any sacrifice time suggesting no exposure to TBHP; in female mice, the number of PE was increased slightly at 48 hr and 72 hr suggesting a weak interaction. These findings are consistent with ADME data which demonstrate negligible systemic distribution of TBHP due to its rapid conversion to 2-methylpropan-2-ol (tert-butanol).