Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March, 1985 - July, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
Well executed and reported study subjected to peer review and conducted according to modern standards, including GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Specifications for the Conduct of Studies to Evaluate the Toxic and Carcinogenic Potential of Chemical, Biological, and Physical Agents in Laboratory Animals for the National Toxicology Program (NTP) October 2006.
Principles of method if other than guideline:
graded oral dosing via gavage. protocol at http://ntp.niehs.nih.gov/go/9987
GLP compliance:
yes
Remarks:
The t-BP studies were performed in compliance with FDA Good Laboratory Practices regulations (21 CFR 58). The Quality Assurance Unit of Battelle Columbus Laboratories performed audits and inspections of protocols, procedures, data, and reports throughout
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
t-BP used in these toxicity studies was manufactured by Penwalt Corporation (Lucidol Division, Buffalo, NY); the
chemical was identified by NMR, infrared, and ultraviolet spectroscopy. Cumulative data derived from iodometric
titration, elemental analysis, HPLC, and thin layer chromatography indicated a purity of > 98.8%. The bulk chemical
was stored at room temperature, protected from light. Quantitative reanalyses were performed; no degradation of
the material was evident.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
F344/N rats used in the 13-week study were produced under strict barrier conditions at Simonsen Laboratories, Inc.
(Gilroy, CA). Animals were progeny of defined, microflora-associated parents that were transferred from isolators to
barrier-maintained rooms. Rats and mice were shipped to the study laboratory at 4 to 5 weeks of age, quarantined
there for 11 days, and placed on study at approximately 6 weeks of age. Blood samples were collected and the
sera analyzed for viral titers from 5 animals per sex and species at study start and termination in the 13-week
studies. Data from 5 viral screens showed that there were no positive antibody titers (Boorman et al., 1986; Rao et
al., 1989). Diet: NIH 07 pelleted feed and water, ad libitum. Animal Room Environment: Temp: 68-75°F; relative
humidity: 35-65%; fluorescent light 12 h/d; 12-15 room air changes/h. Time Held Before Study: 11 d. Age When
Placed on Study: 6 wks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
t-butyl perbenzoate: 0, 30, 60, 125, 250, 500 mg t-butyl perbenzoate per kg body weight in deionized water by
gavage.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
13-weeks
Frequency of treatment:
Duration of Dosing 13-week Studies: 1 x d for 5 d/wk, with two consecutive doses prior to necropsy; last dose
within 24 hrs of necropsy.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 60, 125, 250, 500 mg t-butyl perbenzoate per kg body weight
Basis:
other: as administered via gavage
No. of animals per sex per dose:
10 males; 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Strain and Species: F344/N rats
Animal Source: Simonsen Laboratories, Inc., Gilroy, CA
Chemical Source: Penwalt Corporation, Lucidol Division, Buffalo, NY
Size of Study Groups: 10 males and 10 females per dose group.
Rats were housed 5 per cage.

Method of Animal Distribution: Animals randomized and assigned to study groups using a consecutive identification numbering system.

Time Held Before Study: 11 d
Age When Placed on Study: 6 wks
Duration of Dosing: 1 x d for 5 d/wk, with two consecutive doses prior to necropsy; last dose within 24 hrs of
necropsy.

Age When Killed: 19 wks

Necropsy and Histologic Examinations
Complete necropsies were performed on all animals; organs and tissues were examined for gross lesions. Organs
weighed at the end of the study include brain, forestomach, glandular stomach, spleen, right kidney, testis, thymus,
liver, heart, and lung. Complete examination of all controls and high dose animals; forestomach and gross lesions
examined at lower dose levels. Complete histopathologic examination included the following tissues: gross lesions
and tissue masses (regional lymph nodes), blood smear, mandibular and mesenteric lymph node, salivary gland,
sternebrae, femur, or vertebrae (including marrow), thyroid, parathyroids, liver, gall bladder (mice), heart,
esophagus, stomach (glandular and forestomach), brain (frontal cortex, basal ganglia, pariteal cortex and thalamus,
cerebellum and pons), thymus, pancreas, trachea, small intestine (duodenum, jejunum, ileum), large intestine
(cecum, colon, and rectum), prostate, testes/epididymus, uterus, ovaries, preputial and clitoral glands, lungs and
mainstem bronchi, nasal cavity and turbinates, spleen, kidneys, adrenals, urinary bladder, pituitary, spinal cord and
sciatic nerve (if neurologic symptoms present), eyes (if grossly abnormal), mammary gland (to include surface skin).
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Observed 2 x d for mortality/moribundity; 1 x wk for clinical signs of toxicity; weighed initially, weekly, and at
necropsy.
Sacrifice and pathology:
Animals surviving to the end of the study were killed with CO2. Complete necropsies were performed on all
animals; organs and tissues were examined for gross lesions. Organs weighed at the end of the study include
brain, forestomach, glandular stomach, spleen, right kidney, testis, thymus, liver, heart, and lung.
Complete examination of all controls and high dose animals; forestomach and gross lesions examined at lower
dose levels. Complete histopathologic examination included the following tissues: gross lesions and tissue masses
(regional lymph nodes), blood smear, mandibular and mesenteric lymph node, salivary gland, sternebrae, femur, or
vertebrae (including marrow), thyroid, parathyroids, liver, heart, esophagus, stomach (glandular and forestomach),
brain (frontal cortex, basal ganglia, pariteal cortex and thalamus, cerebellum and pons), thymus, pancreas, trachea,
small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, and rectum), prostate, testes/epididymis,
uterus, ovaries, preputial and clitoral glands, lungs and mainstem bronchi, nasal cavity and turbinates, spleen,
kidneys, adrenals, urinary bladder, pituitary, spinal cord and sciatic nerve (if neurologic symptoms present), eyes (if
grossly abnormal), mammary gland (to include surface skin).Tissues were preserved in 10% neutral buffered
formalin and routinely processed for preparation of histologic sections for microscopic examination. Tissues for
microscopic evaluation were trimmed to a maximum of 3 mm. Following dehydration and embedding, tissues were
sectioned at approximately 5 microns, stained with hematoxylin and eosin, and examined microscopically. Upon
completion of the histologic evaluation by the laboratory pathologist, slides, paraffin blocks, and residual wet tissues
were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. Slides, individual animal data
records, and pathology tables were sent to an independent pathology laboratory for quality assessment; the results
were reviewed and evaluated by NTP’s Pathology Working Group (PWG). The final diagnoses represent a
consensus of contractor pathologists and the PWG. Details of these review procedures have been described by
Maronpot and Boorman (1982) and Boorman et al. (1985).
Statistics:
The significance of differences between dosed and control group means was assessed using multiple comparison
procedures designed to protect against false positive inferences. Either Dunn’s test or Williams’ modification of
Shirley’s multiple comparisons procedure was applied based on the occurrence of a dose-related response in the
data (Dunn, 1964; Shirley, 1977; and Williams, 1986). Shirley’s test is designed to detect treatment-related
differences when the response to treatment consistently increases or decreases as the dose level increases. Dunn’s
test is appropriate if the departure from monotonicity is severe. If the p value from Jonckheere’s test (Hollander and
Wolfe, 1973) for a dose-related trend was greater than or equal
to 0.10, Dunn’s test was used rather than Shirley’s test. The outlier test of Dixon and Massey (1951) was employed
to detect extreme values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
All treated and control male rats survived to the end of the study. One female in the 250 mg/kg group died during
week 5; a control in the female study was removed because it was missexed. Food consumption was similar in all
groups of the treated and control animals except in high dose female rats, whose food consumption was about 7%
less than controls. Body-weight gains of male and female rats in the highest dose groups were depressed after
about week 7.

A variety of clinical observations were noted in both male and female rats during the course of the study, but none
were attributed to administration of t-BP. Similarly, at necropsy, no apparent chemical-related gross lesions were
observed in either sex of rats. Forestomach weights were increased in male rats receiving the 250 and 500 mg/kg
doses and in female rats receiving 60 mg/kg and higher doses. Weights of the glandular stomachs also were
increased in both males and females, but the increases were largely restricted to high dose animals and the
percent increase was smaller than observed in the forestomach. Other changes in organ weights included slightly
decreased spleen weights in males and females receiving the high dose, and increased kidney weights in female
rats receiving 250 mg/kg.

Epithelial hyperplasia and inflammation were observed in the forestomach of dosed rats. Dose-related increases in
the incidence and severity of squamous epithelial hyperplasia were seen in male and female rats. Within the
hyperplastic epithelium there was increased mitotic activity of the basal cell layer, rete peg-like downgrowths of
hyperplastic cells, and variable hyperkeratosis, which appeared to increase in severity with the degree of
hyperplasia present. Inflammatory cell infiltration also was evident in the forestomach of rats in the higher dose
groups. These inflammatory changes included leukocytic exocytosis with neutrophil aggregates within the
hyperkeratotic layer, as well as within intraepithelial clefts and vesicles; congestion of subepithelial capillaries,
perivascular edema, and microhemorrhages were components of inflammation in some rats.

Effect levels

open allclose all
Key result
Dose descriptor:
dose level: 60, 250 and 500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: increased forestomach weight
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Key result
Dose descriptor:
dose level: 500 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: increase in glandular stomach weight
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 30 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: increased forestomach weight/hyperplasia

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral gavage administration of t-BP at doses up to 500 mg/kg produced little or no toxicity past the point of initial
contact, the stomach, characterized with a dose-dependent increase in forestomach weight/hyperplasia in both males and females.
Executive summary:

t-BP was administered by gavage in water to 10 rats of each sex, at doses up to 500 mg/kg. There was a slight depression in food consumption; body weight gains of both sexes in the highest dose group were significantly depressed after week 7. No clinical effects were observed which could be attributed to t-BP administration. Systemic toxicity was not observed. Variations in organ weights were largely restricted to increased stomach weights in both male and female rats. Both the glandular stomach and forestomachs were affected, but the effect on the forestomach was much greater. Hyperplasia of the forestomach mucosa was observed in most groups of dosed rats and increased in severity with dose. Hyperplasia was characterized by increased cellularity and basophilia of the squamous epithelium with variable degrees of hyperkeratosis. Female rats receiving the lowest dose, 30 mg/kg bw/day did not show evidence of forestomach hyperplasia. According to the study report authors, results of this study indicate that oral gavage administration of t-BP at doses up to 500 mg/kg produced little or no toxicity past the point of initial contact, the stomach. Toxicity observed in the stomach, primarily the forestomach, was due probably to the inherent reactivity of t-BP to release free radicals which in turn reacted with the cell membranes of the stomach mucosa. However, the reactivity of t-BP also accounts for its very short half-life in biological systems. Its reaction with stomach contents, stomach tissue, and blood probably prevented t- BP from reaching the systemic circulation and thus accounted for its lack of systemic toxicity. Based on the results presented in this report, it is concluded that the no-observed-adverseeffect- level (NOAEL) for t-BP to induce forestomach lesions in rats and mice is approximately 30 mg/kg.

NOTE: as forestomach effects are not relevant for humans and based on slight spleen and kidney weight changes in the higher dose groups, 125 mg/kg/day was selected as the NOAEL for DNEL derivation.