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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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, acceptable for assessment.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Staatstoezicht op de Volkgezondheid, 19 April 1989; 6 June 1991
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Aqueous solution containing ca. 70wt% TBHP (CAS No. 75-91-2)
- Lot/batch no. 920602/1991

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wigd GmbH, Sulzfeld, Germany
- Age at study initiation: approx. 9-10 wk
- Weight at study initiation: male = 311.1 g; female = 198.5 g
- Housing: individually (during premating, gestation and lactation) or in pairs of one male and one female (during mating)
- Diet (e.g. ad libitum): cereal-based stock diet ad libitum
- Water (e.g. ad libitum): tap-water ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Solutions of test substance were prepared in Millipore-filtered water and stored in brown glass bottles, in a refrigerator, until use.

Concentration in dosing solutions (mg Aq. TBHP-70/ml):
Range finder 1: 0, 10.7, 17.9, 25, 35.7
Range finder 2: 0, 1.25, 2.5, 5
Main study: 0, 0.3, 1, 3

Dosing volume: 10 ml/kg bwt

The test substance was administered once daily by oral gavage (10 ml/kg) with the dose volume adjusted for changes in body weight. Four batches of dosing solutions were prepared for the main study (on June 30, July 14 and 28, and August 11, 1992).
The main study commenced on 1 July 1992 (= nominal day 0) and was terminated on 10-14 August 1992 and the animals sacrificed a few hours after the last dose.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosing solutions were analysed by GC-FID using a Perkin Elmer 8500 gas chromatograph with fused silica 50 m x 0.21 mm column and 0.5 um film methylsilicon PONA. Stability was determined at room temperature for 3 days or in a refrigerator for up to 16 days. Homogeneity was checked by analysing samples from the top, middle and bottom of the dosing solutions. Results demonstrated that the dosing solutions were acceptable for use.
Duration of treatment / exposure:
Range finder 1: 4 days
Range finder 2: 4 days
Main study: 41-45 days
Frequency of treatment:

Daily treatment
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
3, 10 or 30 mg/kg bwt/d
Basis:
nominal in water
Remarks:
Doses / Concentrations:
2.1, 7 or 21 mg/kg bwt/d
Basis:
other: actual dose received
No. of animals per sex per dose:
Range finder 1: 4 males, 4 females
Range finder 2: 2 males, 2 females
Main study: 12 males, 12 females
Control animals:
yes, concurrent vehicle
Details on study design:
The test substance was administered once daily by oral gavage for up to 45 consecutive days.
Positive control:
not relevant

Examinations

Observations and examinations performed and frequency:
Animals were observed for:
- clinical signs: once or twice daily
- body weight: recorded daily
- food consumption: measured weekly
- water consumption: not determined
Sacrifice and pathology:
Necropsy
All survivors were sacrificed by exsanguination from the abdominal aorta under ether anaesthesia and subject to gross necropsy. Samples of the following tissues/organs were preserved in neutral buffered formalin (testes and epididymides in Bouin's fixative):
- ovaries (*) (after counting corpora lutea)
- uterus (after counting implantation sites)
- testes (*)
- epididymides (*)
- seminal vesicles
- liver (*)
- kidneys (*)
- thymus (*)
- adrenals
- brain
- heart
- spleen
- stomach
- organs and tissues showing macroscopic abnormalities
Tissues marked (*) were also weighed.

Histopathology
Tissues from all high dose and control animals were processed and stained with haematoxylin and eosin prior to microscopic examination. Kidneys from intermediate- and low dose males were examined also.
Other examinations:
Haematology: blood (from the abdominal aorta, mixed with EDTA-2K) was examined for:
- haemoglobin
- methaemoglobin
- packed cell volume
- red blood cell count
- reticulocyte count
- total white blood cell count
- differential white blood cell count
- prothrombin time
- thrombocytes
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin (MCH)
- mean corpuscular haemoglobin concentration (MCHC)

Clinical chemistry: blood (from the abdominal aorta, mixed with heparin) was analysed for:
- aspartate aminotransferase activity (ASAT)
- alanine aminotransferase activity (ALAT)
- gamma glutamyl transferase activity (GGT)
- total protein
- albumin
- sodium (Na)
- potassium (K)
- calcium (Ca)
- chloride (Cl) inorganic phosphate cholesterol
- triglycerides
Statistics:
Data were analysed by Fisher's exact test (clinical findings, histopathology), ANOVA (body weight, food consumption, organ weights, haematology data (except as noted below) or ANOVA followed by Dunnett's multiple comparison tests (clinical chemistry data). Methaemoglobin, reticulocyte count and relative differential white blood cell counts were analysed by Kruskal-Wallis nonparametric ANOVA followed by the MannWhitney U-test. Renal changes observed in male rats were analysed by the Cochran-Armitage linear trend test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
2.1 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: multifocal increased accumulation of tubular proteinaceous material and tubular nephrosis
Dose descriptor:
NOAEL
Effect level:
21 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: with the exception of renal changes, no adverse effects at highest dose tested
Dose descriptor:
NOAEL
Effect level:
21 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: no adverse effects at highest dose tested
Basis for effect level:
other: Remark: toxicokinetic data demonstrate rapid conversion of TBHP to 2-methylpropan-2-ol (tert-butanol) in vivo, thereby minimising any potential for uptake and systemic distribution of unchanged TBHP following oral exposure.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Stability data

Aqueous solutions containing 0.3, 2.5 or 25 mg/ml indicated that the test substance was stable in water when stored at room temperature for three days or in a refrigerator for up to 16 days. Concentrations of test substance present in samples taken at the top, middle and bottom of the flask indicated that this substance was homogeneously distributed in the dosing solutions at all dose levels, with achieved concentrations very close to the target concentrations (coefficient of variation 0.34-1.2%).

Survival and clinical signs

There were no substance-related deaths or clinical signs, although one low dose female and one intermediate dose female died prior to scheduled necropsy due to dosing errors.

Body weight, body weight gain and food consumption

There were no treatment-related or statistically significant alterations in mean body weight, body weight gain or food consumption.

Haematology

With the exception of a significant increase in absolute (but not relative) monocyte count in high dose females (control: 0.00  0.00 x 10^9/l; 30 mg/kg bwt/d: 0.04  0.01 x 10^9/l; P<0.05), total and differential white cell counts were unaffected by treatment. This finding was considered of no toxicological relevance given the low control value and the absence of other alterations in white cell parameters.

The only treatment-related alteration in red cell counts was a statistically significantly decrease in reticulocyte counts in high dose males (control: 19.0 per 1000; 30 mg/kg bwt/d: 10.7 per 1000; P<0.01).

All remaining haematological parameters were unremarkable.

Clinical chemistry

Findings were limited to dose-related alterations in total bilirubin concentration, which were increased in males but decreased in females:

Males: 0, 3, 10 or 30 mg/kg bwt/d = 0.98, 1.48, 2.02** or 2.12** umol/l, respectively

Females: 0, 3, 10 or 30 mg/kg bwt/d = 1.85, 2.10, 0.99*, 0.82** umol/l, respectively

Values presented as mean; * = P<0.05, ** = P<0.01

The inconsistent nature of these changes suggests they are of doubtful toxicological relevance.

Organ weights

With the exception of a slight (7%) statistically significant increase in relative kidney weight in intermediate level males, organ weight data were unremarkable.

Gross necropsy

No treatment-related macroscopic changes were detected at autopsy. (Comment: Findings at gross necropsy from the range-finding study using higher treatment levels were limited to moderate (all animals given 107 mg/kg bwt/d) or severe (all animals given 179 mg/kg bwt/d and above) submucosal oedema of the stomach wall affecting both sexes; no effect at 50 mg/kg bwt/d for 5 days).

Histopathology

Microscopic examination revealed treatment-related changes in the kidneys of male rats, characterised by multifocal increased accumulation of tubular proteinaceous material (e.g. occurrence of rounded intracytoplasmatic hyaline inclusions) and tubular nephrosis (including pyknotic nuclei in the proximal tubular cells). Similar renal lesions were present in a few control males however the incidence and severity of the findings in treated animals appeared dose-related:

multifocal accumulation, proteinaceous material: 4/12, 6/12, 9/12, 10/12*, respectively

tubular nephrosis: 4/12, 5/12, 9/12, 10/12*, respectively

* = P<0.05

The NOAEL for these findings was 3 mg/kg bwt/d.

Changes in other organs examined were typical for rats of the strain and age.

Applicant's summary and conclusion

Conclusions:
No evidence from this study of systemic toxicity following repeated oral exposure.
Executive summary:

The repeated dose toxicity of TBHP was investigated in a GLP-compliant guideline combined repeated dose and reproductive/developmental toxicity screening test (OECD 422) in which groups of male and female Wistar rats (12/sex/dose level) received TBHP by oral gavage in water at nominal doses of 0, 3, 10 or 30 mg/kg bwt/day for 45 consecutive days (actual received doses = 0, 2.1, 7 or 21 mg/kg bwt/day). Dose levels were based on results obtained from two preliminary range-finding studies which demonstrated moderate to severe damage to the stomach lining at higher dose levels. A dose-related increase in the incidence of renal lesions (described by the study pathologist as multifocal increased accumulation of tubular proteinaceous material and multifocal tubular nephrosis) was observed in males at all doses (females unaffected). There were no other findings of note. The study pathologist concluded that the kidney lesions resembled those associated with alpha-2u-globulin nepthropathy, however a retrospective, independent expert histological evaluation of kidney tissue did not confirm this finding (Hard, 2007). The NOAEL for systemic toxicity (both sexes) from this study was therefore 21 mg TBHP /kg bwt/d, the highest dose tested.