tert-butyl perbenzoate

Endpoint summary

Description of key information

14-day (dose levels 70, 140, 278, 556 and 1112 mg/kg) and 13-week (dose levels 0, 30, 60, 125, 250 and 500 mg/kg) studies in both mice and rat (NTP, 1992) resulted to a NOEAL of 30 mg/kg for forestomach lesions. Systemic toxicity was not observed in either species with oral doses as high as 1112 mg.

In the 13-week study, only observed effects at study termination were slightly lower thymus and spleen weights (absolute and relative to BW) in males at the highest dose level of 500 mg/kg. Other variations in organ weights observed in male and female rats appeared neither remarkable nor dose-related. Histopathological examination revealed no lesions that were considered related to administration of t-BP.

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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.

Qualifier:

according to guideline

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

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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

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.

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

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.

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 thymus and spleen weights (absolute and relative to BW) in males at the highest dose level of 500 mg/kg, and an increased kidney weights in female rats only at 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.

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

Critical effects observed:

not specified

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 of 0, 30, 60, 125, 250 and 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. No specific signs of systemic toxicity were observed, indicating a NOAEL of at least 500 mg/kg bw/day.

 

Reference 2

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.

Qualifier:

according to guideline

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

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

B6C3F1 mice 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 at study start and termination in the 13-week studies. Data from 12 viral screens performed in mice 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

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.

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: B6C3F1 mice
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.
Mice 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

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, gall bladder, 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).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).

Other examinations:

At the end of the 13-week study, blood smears were prepared from mice for erythrocyte micronuclei determinations. Blood smearswere prepared (unstained) and fixed in 100% methanol.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

Two mice died during the study. One control male died on day 4 of apparent gavage error, and a female in the 250 mg/kg dose group died on day 3. Mean diet consumption and body-weight gains by all dosed groups of mice were similar to those of the respective control groups. Clinical observations during the course of the study and gross observations at necropsy revealed few signs of toxicity or macroscopic lesions related to t-BP administration. Evidence of t-BP toxicity in mice was limited to increased stomach weights and lesions in the stomachs of dosed animals. Forestomach weights of both sexes receiving 250 and 500 mg/kg were increased by 50% or more. Additionally, glandular stomach weights of female mice in the 500 mg/kg group, and the glandular stomach-to-body-weight ratios of female mice in the 250 mg/kg group were significantly increased compared to controls. Other changes in organ weights and organ-to-body-weight ratios were not considered related to chemical toxicity. Histopathological examination of the forestomachs revealed compoundrelated hyperplasia of the stratified squamous epithelium in male and female mice. In males, all dose groups were affected, although hyperplasia in a single animal in the 30 mg/kg group was minimal and not clearly compound-related. In females, the lesion was seen in the 60 mg/kg and higher dose groups. This lesion increased in both frequency and severity as the dose increased. Hyperplasia was characterized by increased cellularity and basophilia of the squamous epithelium, with hyperkeratosis that also appeared to increase with dose.

Key result

Dose descriptor:

dose level: 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:

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:

other: 250 mg/kg bw/day

Sex:

female

Basis for effect level:

other: increased glandular stomach weight-to-body-weight ratio

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

Critical effects observed:

not specified

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, characterizedwith a dose-dependent increase in forestomach weight/hyperplasia in both males and females.

Note: Forestomach effects are not considered relevant for human hazard assessment.

Executive summary:

t-BP was administered by gavage in water to 10 mice of each sex, at doses up to 500 mg/kg. t-BP toxicity observed in mice was limited to increased forestomach weight in most dose groups and to less dramatic increases in glandular stomach weight in mice receiving the highest doses. Forestomach toxicity was characterized by dose-dependent increases in hyperplasia of the squamous epithelium in all mice except those in the low dose group. Systemic toxicity was not observed.

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-adverse-effect- level (NOAEL) for t-BP to induce forestomach lesions in rats and mice is approximately 30 mg/kg.

Reference 3

Endpoint:

short-term repeated dose 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.

Qualifier:

according to guideline

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

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

B6C3F1 mice used in the 14-day 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. 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. 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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

t-butyl perbenzoate: 0, 70, 140, 278, 556, 1112 mg per kg body weight in corn oil, by gavage.

As these studies were carried out in a manner to also evaluate the effect of equimolar doses of the normal degredation products of t-BP in biological media, separate groups of animals were also dosed as follows: 30, 60, 120, 242, 484 mg t-butanol; or 40, 80, 160, 321, 642 mg benzoic acid per kg body weight in corn oil, by gavage.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

Duration of Dosing 14-Day Studies: 1 x d for 5 d/wk for total of 12 doses over 16 days.

Remarks:

Doses / Concentrations:
0, 70, 140, 278, 556, 1112 mg per kg body weight in corn oil, by gavage.
Basis:
other: as administered via gavage

No. of animals per sex per dose:

5 males; 5 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Strain and Species: B6C3F1 mice
Animal Source: Simonsen Laboratories, Inc., Gilroy, CA
Chemical Source: Penwalt Corporation, Lucidol Division, Buffalo, NY
Size of Study Groups: 5 males and 5 females of each species per dose group.
Mice 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: 14-Day Studies: 1 x d for 5 d/wk for total of 12 doses over 16 days.

Age When Killed: 14-Day Studies: 8 wks

Necropsy and Histologic Examinations
Necropsy performed on all animals; the following tissues were examined microscopically: complete examination of controls and high dose animals; stomach, esophagus, urinary bladder, and right kidney examined at all lower doses.

Positive control:

none

Observations and examinations performed and frequency:

Observed 2 x d for mortality/moribundity; 1 x wk for clinical signs of toxicity; weighed initially, on day 8, 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. 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. The specific tissues examined:complete examination of controls and high dose animals; stomach, esophagus, urinary bladder, and right kidney examined at all lower doses. Organs weighed at the end of the study include brain, forestomach, glandular stomach, spleen, right kidney, testis, thymus, liver, heart, and lung.

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.

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

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:

T-butyl peroxybenzoate
No male mice receiving t-BP died during the study. One female mouse receiving the highest dose of t-BP was killed in moribund condition on study day 3. Weight gains of groups of treated male and female mice were generally similar to those of controls. Mice dosed with t-BP showed no clinical signs considered to be related to administration of the chemical. The only lesion observed at necropsy that was considered possibly treatment-related was a single pigmented focus in the stomach of a female mouse receiving the highest dose of t-BP. Similarly, little or no effect of administration of t-BP was observed on most absolute or relative organ weights. However, stomach weights were increased by as much as 2-fold, in a dose-dependent manner, in the 3 highest dose groups of male mice and in the 2 highest dose groups of female mice. Lesions considered related to administration of t-BP were observed only in the forestomach. These lesions included hyperplasia, ulceration, and acute inflammation of the forestomach mucosa. Hyperplasia was the most common lesion and was characterized by increased cellularity and basophillia of the epithelium, with variable degrees of hyperkeratosis. Hyperplasia was observed in the forestomachs of all mice that received the 2 highest doses and which survived the 14-day study, and in 3 of 5 females receiving the third highest dose, 278 mg/kg. A shallow ulcer was observed in the forestomach of 1 female mouse receiving the highest dose, corresponding to the gross lesion mentioned above.

Comparative results with t-Butanol and Benzoic acid:
No male mice receiving t-butanol died during the study. However, 3 males receiving the highest dose of benzoic acid died during week 1. Another female, receiving the highest dose of t-butanol, died on day 4; and a female receiving the highest dose of benzoic acid was killed in moribund condition midway through the study. Food consumption was somewhat lower in groups of mice of both sexes receiving t-butanol and benzoic acid, but differences were not significant in either sex compared to controls. Mice dosed with t-butanol showed no clinical signs considered to be related to administration of the chemical. However, male and female mice receiving the 642 mg/kg dose of benzoic acid exhibited rough hair coats, labored breathing, hunched posture, salivation, and enlarged abdomen. As indicated, 3 of 5 male mice in this benzoic acid dose group died during week 1. Increases in stomach weights were observed in mice receiving t-butanol, but these were not dose-related; no increases in stomach weights were observed in mice receiving benzoic acid.

Dose descriptor:

dose level: 1112, 556, 278 mg/kg bw

Sex:

male

Basis for effect level:

other: increased stomach weight

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

dose level: 1112, 556 mg/kg bw

Sex:

female

Basis for effect level:

other: increased stomach weight

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

dose level: 1112, 556, 278 mg/kg bw

Sex:

male/female

Basis for effect level:

other: hyperplasia of forestomach characterized by increased cellularity and basophillia of the epithelium, with variable degrees of hyperkeratosis

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Critical effects observed:

not specified

Conclusions:

Adverse effects of t-BP, in this study, were limited to hyperplasia of the forestomach, along with increased absolute and relative stomach weights in male and female mice. Consistent with this, one shallow ulcer of the forestomach was found in one female in the high-dose group. No effect on mortality was observed.

An equimolar dose of benzoic acid demonstrated greater mobidity and mortality, while t-butanol only resulted in slight [but not significant] reduction in food consumption.

Executive summary:

Results of 14-day toxicity studies with 5 animals of each sex of mice indicated that t- BP, adminstered by gavage in corn oil in doses ranging from 70 to 1112 mg/kg, produced no marked signs of systemic toxicity. Toxicity in mice, attributable to t-BP, was limited largely to increased stomach weights in males and females receiving the highest doses. This toxicity was characterized by forestomach epithelial hyperplasia, ulceration, and acute inflammation. Equimolar doses of the degradation products of t-BP (t-butanol and benzoic acid) also were administered in the 14-day studies to determine if t-BP toxicity could be attributed to the parent compound or products of its chemical degradation and/or metabolism. Results of these studies indicated that equimolar doses of t-butanol were not toxic in either sex or species. Some systemic toxicity of benzoic acid was observed in both sexes of mice receiving the highest dose. Toxicity was evidenced by the poor condition of dosed animals and in several deaths during the first week of the study. No lesions were observed microscopically in mice dosed with benzoic acid. It is speculated that this systemic toxicity may have been due to acidosis.

Reference 4

Endpoint:

short-term repeated dose 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.

Qualifier:

according to guideline

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

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Fischer 344 rats used in the 14-day 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 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. 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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

t-butyl perbenzoate: 0, 70, 140, 278, 556, 1112 mg per kg body weight in corn oil, by gavage.

As these studies were carried out in a manner to also evaluate the effect of equimolar doses of the normal
degradation products of t-BP in biological media, separate groups of animals were also dosed as follows: 30, 60,
120, 242, 484 mg t-butanol; or 40, 80, 160, 321, 642 mg benzoic acid per kg body weight in corn oil, by gavage.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

14 days

Frequency of treatment:

Duration of Dosing 14-Day Studies: 1 x d for 5 d/wk for total of 12 doses over 16 days.

Remarks:

Doses / Concentrations:
0, 70, 140, 278, 556, 1112 mg per kg body weight in corn oil, by gavage.
Basis:
other:

No. of animals per sex per dose:

5 males; 5 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: 5 males and 5 females of each species 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: 14-Day Studies: 1 x d for 5 d/wk for total of 12 doses over 16 days.
Age When Killed: 8 wks
Necropsy and Histologic Examinations
Necropsy performed on all animals; the following tissues were examined microscopically: complete examination of
controls and 3 highest doses of benzoic acid, and highest dose groups for t-BP and t-butanol; stomach, esophagus,
urinary bladder, and right kidney examined in all other dose groups.

Positive control:

none

Observations and examinations performed and frequency:

Observed 2 x d for mortality/moribundity; 1 x wk for clinical signs of toxicity; weighed initially, on day 8, 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. 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. The
specific tissues examined:complete examination of controls and 3 highest doses of benzoic acid, and highest dose
groups for t-BP and t-butanol; stomach, esophagus, urinary bladder, and right kidney examined in all other dose
groups. Organs weighed at the end of the study include brain, forestomach, glandular stomach, spleen, right
kidney, testis, thymus, liver, heart, and lung.

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.

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

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

T-butyl peroxybenzoate
No deaths occurred among control rats, or among rats that received t-BP. Food consumption by dosed males and
females was comparable to or slightly higher than that of the control groups. Body-weight gains of all treated male
rats, except those in the high dose t-BP group, were greater than those of the control group. However, the low
weight gain of the control group of male rats may account for these findings. Female rats receiving the highest
dose of t-BP gained less weight than controls, but the difference was not statistically significant. No treatmentrelated
gross lesions were observed in rats at the end of the studies. At study termination, liver weights of female
rats receiving 556 and 278 mg/kg t-BP were higher by as much as 20%, than those of controls; both these groups,
and females in the high dose group (1112 mg/kg), had higher mean liver-to-body-weight ratios. Thymus weights
and thymus-to-body- weight ratios of female rats in the highest dose t-BP group were about 20% lower than
controls. Stomach was considered a possible target tissue for t-BP administrated by gavage, but the only increases
in absolute and relative stomach weight (about 25%) were seen with male rats receiving the highest dose. Other
variations in organ weights observed in male and female rats appeared neither remarkable nor dose-related.
Histopathological examination revealed no lesions that were considered related to administration of t-BP

Comparative results with t-Butanol and Benzoic acid:
No deaths occurred among control rats, or among rats that received t-butanol. Two male rats receiving benzoic
acid died during the study. One receiving 321 mg/kg (benzoic acid) was killed in a moribund condition on day 6;
the other, in the 160 mg/kg group (benzoic acid), died on day 13. Female rats receiving the highest dose of
benzoic acid gained less weight than controls, but the difference was not statistically significant. During the in life
portion of this study, 1 male rat receiving 321 mg/kg of benzoic acid was observed to have labored respiration and
was lethargic, leading to its moribund sacrifice. Histopathological examination revealed no lesions that were
considered related to administration of t-butanol, or benzoic acid.

Dose descriptor:

dose level: 1112 mg/kg bw (t-BP)

Sex:

male

Basis for effect level:

other: increased stomach weight

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

dose level: 278 and 556 mg/kg bw (t-BP)

Sex:

female

Basis for effect level:

other: increased liver weight

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Dose descriptor:

dose level: 1112 mg/kg bw (t-BP)

Sex:

female

Basis for effect level:

other: decreased thymus weight

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Critical effects observed:

not specified

Conclusions:

Signs of toxicity observed in rats were largely limited to increased stomach weights in males receiving the highest
dose of t-BP. Equimolar doses of t-butanol and benzoic acid were not toxic to rats under these conditions of
exposure.

Executive summary:

Results of 14-day toxicity studies with 5 animals of each sex of rats indicated that t- BP, administered by gavage in corn oil in doses ranging from 70 to 1112 mg/kg, produced no marked signs of systemic toxicity. Toxicity in rats, attributable to t-BP, were largely limited to increased stomach weights in males receiving the highest dose of t-BP. Equimolar doses of the degradation products of t-BP (t-butanol and benzoic acid) also were administered in the 14-day studies to determine if t-BP toxicity could be attributed to the parent compound or products of its chemical degradation and/or metabolism. Results of these studies indicated that equimolar doses of t-butanol and benzoic acid were not toxic to rats under these conditions of exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Well conducted studies, by the National Toxicology Program, in rats and mice.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The extensive studies performed by NTP have shown that tert-butyl peroxybenzoate is rapidly hydrolysed into t-butanol and benzoic acid, mostly in gastro-intestinal tract, and possible remainder of the substance is even more rapidly hydrolysed after absorption with t½ of 4 minutes in blood. Consequently, tert-butyl peroxybenzoate cannot be expected to be systemically available in significant amounts, and systemic toxicity is expected to be characterized by the hydrolysis products t-butanol and benzoic acid. The hydrolysis products are rapidly excreted.

Additional information

Extensive studies in mice and rat have been performed by NTP:

Rats: 14-Day: Results of 14-day toxicity studies with 5 animals of each sex of rats indicated that t-BP, administered by gavage in corn oil in doses ranging from 70 to 1112 mg/kg, produced no marked signs of systemic toxicity. Toxicity in rats, attributable to t-BP, was largely limited to increased stomach weights in males receiving the highest dose of t-BP.

13-Week: 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. NOAEL ~30mg/kg, based on forestomach effects.

Mice: 14-Day: Results of 14-day toxicity studies with 5 animals of each sex of mice indicated that t- BP, administered by gavage in corn oil in doses ranging from 70 to 1112 mg/kg, produced no marked signs of systemic toxicity. Toxicity in mice, attributable to t-BP, was limited largely to increased stomach weights in males and females receiving the highest doses. This toxicity was characterized by forestomach epithelial hyperplasia, ulceration, and acute inflammation.

13-Week: t-BP was administered by gavage in water to 10 mice of each sex, at doses up to 500 mg/kg. t-BP toxicity observed in mice was limited to increased forestomach weight in most dose groups and to less dramatic increases in glandular stomach weight in mice receiving the highest doses. Forestomach toxicity was characterized by dose-dependent increases in hyperplasia of the squamous epithelium in all mice except those in the low dose group. Systemic toxicity was not observed.

Based on the results presented in this report, it is concluded that the no-observed-adverse effect-level (NOAEL) for t-BP to induce forestomach lesions in rats and mice is approximately 30 mg/kg. As no signs of systemic toxicity were observed, the NOAEL for systemic toxicity was considered to be at least 500 mg/kg/day.

 

The substance has been evaluated by Italy under CoRAP. The Substance Evaluation Conclusion document from 2020 stated:

“Due to the 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, the NOAEL in this study is identificated by eMSCA is 125 mg/kg bw/day since the effects on foresthomach at 60 mg/kg bw/day and higher doses in females are not considered relevant for human.”
“In these studies, as well as in the other repeated studies presented in the dossier, toxicological effects in organs are not considered to fulfil the CLP criteria for classification as target organ toxicity, repeated exposure, as forestomach effects are considered not relevant for humans and based on slight spleen and kidney weight changes in the higher dose groups in the 13 week study on F344 rats, 125 mg/kg/day was selected as the NOAEL for DNEL derivation.”

The attached picture shows the absolute and relative organ weights from the 13-week study in rats for spleen and kidney. The lower relative spleen weight is only significant for the males at the high dose of 500 mg/kg. The absolute (kidney weight is less affected by body weight) higher kidney weight is significant in females only at 250 mg dose group, and not at the higher 500 mg/kg dose. As such this finding is likely not treatment related.
The use of 125 mg as starting point for DNEL derivation therefore is overly conservative.

Justification for classification or non-classification

Classification for STOT-RE Cat. 2 is required in case of significant toxic effects at levels ≤ 100 mg/kgbw/d.
The available 90-day studies in rat and mice only indicate local effects in forestomach visible from dose levels of 60 mg/kg bw. However, these effects are not relevant for humans. The studies do further not give indication of other effects that suggest severe toxicity at levels requiring consideration for classification for STOTS-RE.