Di-tert-butyl peroxide

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study was conducted using a non-guideline multistage model of carcinogenesis using mouse skin.

Qualifier:

no guideline followed

Principles of method if other than guideline:

multistage model of carcinogenesis using mouse skin

GLP compliance:

not specified

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Species:

mouse

Strain:

Sencar

Sex:

female

Details on test animals or test system and environmental conditions:

Female virgin outbred SENCAR mice (5±6 weeks old) were purchased from
the National Cancer Institute Laboratories (Frederick, MD) and housed by
treatment group, up to 10 mice/cage. Temperature was maintained at 22
4C, with a relative humidity of 50 20%. Temperature and humidity were
recorded at least once daily. Controls were set to maintain a 12 h light/12 h
dark cycle. There were 12 or more air changes per hour in the room where the
study animals were housed. The experimental animal facility was accredited by the
American Association for Assessment and Accreditation of Laboratory Animals
and caging complied with Institutional Animal Care and Use Committee
requirements ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004)

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

Mouse weight was used to randomly assign
animals to treatment groups. Upon group assignment, the weight variation of
animals did not exceed 2 SD from the mean body weights and the mean body
weights for each treatment group were not statistically different. Animals (7±9
weeks old) entered into the study were shaved with surgical clippers at least
2 days before. Only those in the resting phase of the hair cycle, i.e. animals that
did not show any hair regrowth, were used in the study. Peroxides and control
chemicals were applied to the interscapular/lumbar region of the back in a total
volume of 200 ml. ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004)

Duration of treatment / exposure:

See details on study design

Frequency of treatment:

twice per day for 4 weeks

Remarks:

Doses / Concentrations:
100 and 200 umol
Basis:
nominal conc.

No. of animals per sex per dose:

5 per treatment

Details on study design:

4 week study: The test article was evaluated for its ability to induce sustained epidermal proliferation, dermal inflammation and oxidative damage of epidermal DNA when applied topically for 4 weeks. Specimens of dosed skin were harvested 2 and/or 4 days after final dosing and increases in the mentioned parameters were compared with those produced by the appropriate vehicle and positive (100 nmol DMBA or 2 mg TPA) controls.

DMBA, 7,12- dimethylbenz[a]anthracene
TPA, 12-O-tetradecanoylphorbol-13-acetate

Examinations:

Cutaneous effects related to carcinogenic
potential, namely effects on sustained epidermal hyperplasia,
dermal inflammation, oxidative damage to skin DNA,
and if above parameters significantly affected, mutation of codon 12, 13 or 61 of the c-Ha-ras protooncogene.

Details on results:

DTBP had no effect on the three biomarkers of tumor promotion in mouse skin, i.e. sustanied epidermal hyperplasia, dermal inflammation and oxidative DNA damage. Therefore, further examination of DTBP's ability to produce mutation in codons 12, 13, and 61 of the c-Ha-ras protooncogene, i.e. those mutation known to be involved in the initiation of mouse skin tumors, was not performed. ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004)

Conclusions:

The authors concluded that, DTBP had no effect on the three biomarkers of tumor promotion in mouse skin, i.e. sustanied epidermal hyperplasia, dermal inflammation and oxidative DNA damage. Therefore, further examination of DTBP's ability to produce mutation in codons 12, 13, and 61 of the c-Ha-ras protooncogene, i.e. those mutation known to be involved in the initiation of mouse skin tumors, was not performed. ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004)

Executive summary:

A multistage model of carcinogenesis using mouse skin was used to evaluate the carcinogenic potential of DTBP. DTBP was tested for its ability to increase biomarkers of tumor promotion in mouse skin, i.e. sustained epidermal hyperplasia, dermal inflammation and oxidative DNA damage using SENCAR mice exposed topically for 4 weeks. DTBP did not exhibit significant increases in all three biomarkers associated with tumor promoting activity.

Reference 2

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-guideline study to determine the skin tumor promoting potential; non-GLP

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two-stage initiation promotion. Mice were initiated with a single topical application of 10 nmol DMBA (7,12-dimethylbenz(a)anthracene) followed 2 weeks later by twice-weekly application of the test substance.

GLP compliance:

not specified

Type of method:

in vivo

Endpoint addressed:

other: tumor promotion

Species:

mouse

Strain:

Sencar

Sex:

female

Details on test animals or test system and environmental conditions:

Animal treatment. Female SENCAR mice were obtained from the NCI
(Frederick, MD) and were maintained in a temperature- and humidity-controlled
room on a 12-h light/dark cycle, 10 per polycarbonate cage on corn cob
bedding. Two days before initiation, when the mice were 7–9 weeks old, the
dorsal skin was clipped free of hair with surgical clippers, and only those mice
in the resting phase of the hair cycle were used. Each experimental group
consisted of 30 mice. [Gimenez-Conti et al TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 73–79 (1998)]

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

All solutions of DMBA,were prepared in reagent-grade
acetone immediately before use and were applied topically in a total volume of
0.2 ml. Mice were initiated with a single topical application of 10 nmol
DMBA, followed 2 weeks later by twice-weekly applications of the different
peroxides. Control groups were included in which the mice received acetone in
place of DMBA and then were subsequently treated with the peroxides. For all
groups, peroxide treatments were for 60 weeks, (10, 20, 30 and 60 mg) [Gimenez-Conti et al TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 73–79 (1998)]

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

See above

Frequency of treatment:

See above

Remarks:

Doses / Concentrations:

Basis:

No. of animals per sex per dose:

30

Control animals:

yes, concurrent vehicle

Details on study design:

The number and incidence of skin tumors (.2 mm) were recorded weekly.
At the end of the experiment, all the mice were killed, and all skin tumors were
routinely fixed and processed for histological analysis. The diagnosis of skin
tumors was confirmed histologically. For groups in which tumors were observed,
the weekly number of papillomas per mouse was calculated by dividing
the total number of papillomas observed on surviving mice by the number of
surviving mice.

Statistical methods. The incidences of papillomas and squamous cell
carcinomas (SCC) were subjected to Kaplan–Meier survival analysis (Lee,
1992) (Statistica, Statsoft, Tulsa, OK). The time in weeks to the incidence of
the first tumor (either papilloma or squamous cell carcinoma) was noted along
with the animals that became unevaluable or did not develop a tumor. This data
was used in the Kaplan–Meier analysis. Tumor multiplicity data at maximum
was analyzed using analysis of variance and the Kruskal–Wallis one-way
analysis of variance by ranks using SPSS (Zar, 1996) (SPSS Inc., Chicago, IL).
The Kruskal–Wallis was more appropriate as the counts of papillomas were
discrete and near zero for several groups. [Gimenez-Conti et al TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 73–79 (1998)]

Positive control:

Tumor incidence was compared to the group receiving DMBA as the initiator and benzoyl peroxide as the promoter.

Details on results:

Di-tert-butyl peroxide did not induce tumor formation on initiated skin.

Conclusions:

Di-tert-butyl peroxide, was totally inactive in promoting papillomas or carcinomas in initiated skin. [Gimenez-Conti et al TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 73–79 (1998)]

Executive summary:

The study was conducted to evaluate the skin tumor-promoting potential of some organic peroxides including di-tert-butyl peroxide in the two-stage initiation promotion study. Female SENCAR mice were initiated dermally with DMBA followed by di-tert-butyl peroxide. The number and incidence of skin tumors were determined after 60 weeks.

Di-tert-butyl peroxide, was totally inactive in promoting papillomas or carcinomas in initiated skin. [Gimenez-Conti et al TOXICOLOGY AND APPLIED PHARMACOLOGY 149, 73–79 (1998)]

Reference 3

Endpoint:

specific investigations: other studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Non-guideline study, non-GLP with limited study details and data (i.e. no histopathology data).

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two-stage mechanism of skin carcinogenesis

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

carcinogenicity

Species:

mouse

Strain:

Swiss

Sex:

female

Details on test animals or test system and environmental conditions:

This colony originated from Swiss mice obtained in 1951 from
the Roscoe B. Jackson Memorial Laboratory, Bur Harbor, Maine, and has
since been bred randomly in this laboratory. The mice were kept in
plastic cages with wood shavings and fed Rockland mouse diet and tap
water ad libitum. A standard technique was used in all the tests with the
exceptions that will be described. All substances were applied with a glass
dropper on an area of skin approximately 2 X 2 cm in the interscupular
region that had been clipped free of hair before the first application and
subsequently kept free of hair by periodic clipping with scissors.
(Saffiotti et al Natl. Cancer Inst. Monogr. 10: 489-507, 1963)

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

Di-t-butyl peroxide, 0.5% in acetone, was applied twice weekly for 3 weeks
followed 1 week later by the promoting agent, Croton oil, 5% in mineral oil twice weekly
for 80 weeks or di-t-butyl peroxide, 0.5% in acetone, twice weekly for 81 weeks.

All aninlais were checked twice weekly, and skin lesions were charted weekly
on graph paper. All mice were observed until spontaneous death or
killed when dying. All tumor-bearing mice and many apparently tumor free,
with the exception of some that were lost through cannibalism, were
examined histologically. Experimental treatments and results are reported
in the tables that record the total number of skin-tumor-bearing animals
and the total number of tumors observed. Skin lesions that persisted less
than 4 weeks were not counted as tumors. Some tumors were found to be
malignant either grossly or microscopically, and they are listed as carcinomas.
The other tumors were all papillomas of the various types previously
described. Some papillomas were observed grossly and found to
regress and are listed as regressions. The average latent period for the
appearance of all the tumors is also recorded. (Saffiotti et al Natl. Cancer Inst. Monogr. 10: 489-507, 1963)

Details on analytical verification of doses or concentrations:

None provided

Duration of treatment / exposure:

See above

Frequency of treatment:

See above

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Examinations:

See above

Conclusions:

Di-t-butyl peroxide did not exhibit initiating power in a two-stage skin carcinogenesis study (Saffiotti et al Natl. Cancer Inst. Monogr. 10: 489-507, 1963).

Reference 4

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-guideline study to study mechanism of promotion; non-GLP; no statistical analysis

Qualifier:

no guideline followed

Principles of method if other than guideline:

The parameters evaluated in this study
include a series of short-term markers of tumor promotion,
hyperplasia, induction of dark basal keratinocytes and
induction of ornithine decarboxylase activity. (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991)

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

other: mechanism of tumor promotion

Species:

mouse

Strain:

Sencar

Sex:

female

Details on test animals or test system and environmental conditions:

None provided

Route of administration:

dermal

Vehicle:

acetone

Details on exposure:

Animals and treatmemt
Female Sencar mice (1-9 weeks;) were shaved with surgical clippers 2 days before
treatment and only those in the resting phase of the hair cycle were used. In the
first experiment the animals were treated topically once with any one of the seven
compounds in acetone solution or acetone alone. For each compound there were
three groups of mice receiving different doses. Following a single application
on the dorsal skin, four or five anirna1s were killed 0.25, I, 2, 4 or 6 days
after treatment. In the second experiment, each animal was treated topically twice
weekly for 2 weeks with one of the eight compounds in acetone solution or with
acetone alone. As above, three different dose Ievels of the compounds were studied.
Groups of four to five animals were killed at 0.25, 1,2,4 or 7 days after the
last application. (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

See above

Frequency of treatment:

See above

Remarks:

Doses / Concentrations:

Basis:

No. of animals per sex per dose:

See above

Control animals:

yes

Details on study design:

ODC (ornithine decarboxylase) determinations
To obtain sufficient material, the dorsal skins from each of four to five mice
were pooled to constitute each treament group. The mice were killed by cervical
dislocation and excess hair removed by a 2 min Nudit/Nair application, skin was
washed extensively and the treated area removed. After excision of 1 cm2 of
treated skin for the study of hyperplasia and dark cells, the epidermis was obtained
by the cold scraping method previously described. Briefly, skin was placed
epidermis side down on an ice-cold glass plate and excess fat am loose connective
tissue were removed by scraping with a scalpel. Then the skin was turned around
with the epithelium face up and the epidermis was scraped with a scalpel blade.
Periodically histological evaluation of the scraped skin was performed to monitor
this procedure. Tissue was homogenized (Polytron) in a mixture containing sodium
potassium phosphate (50 mM), pH 7, pyridoxal phosphate (0.2 mM) and EDTA
(0.1 mM) and processed as described previously by Weeks and Slaga.

Morphometric studies and quantitative evaluation of dark cells.
Half the excised treated skin was fixed in neutral formalin, embedded in paraffm
and stained with hematoxylin and eosin, and the other half was flxed in 3 %
glutaraIdehyde in 0.05 M symcollidine buffer (PH 7.4), postfixed in 2% osmium
tetroxide, embedded in Epon 12 and stained with toluidine blue according to
techniques previously reported.
The epithelial thickness was measured randomIy at 20 different sites of
interfollicular epidermis in each animal in paraffin-embedded blocks.
Dark basal keratinocytes and basal cells in the interfollicular epidermis were
counted (I block/animal, four to five animals/dose/time point). One thousand
cells were counted for each dose and time point. (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991)

Examinations:

See above

Positive control:

No. Results were compared to benzoyl peroxide (BzPo).

Details on results:

Di-tert-butyl peroxide (DTBP) stimulated ODC activity. DTBP induced less ODC activity than a singel dose of BzPo. In mice that received multiple applications, DTBP induced low ODC activity.

After a single applicaton DTBP induced non-dose response, transient epidermal hyperplasia (slight inflammatory alteration in the dermis) when compared to the control acetone. DTBP showed no hyperplastic effect at any dose level following mutiple treatments. DTBP did not increase the percentage of dark basal keratinocytes compared to acetone. (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991)

Conclusions:

After a single applicaton, DTBP induced non-dose response, transient epidermal hyperplasia (slight inflammatory alteration in the dermis) when compared to the control acetone. DTBP showed no hyperplastic effect at any dose level following mutiple treatments. DTBP did not increase the percentage of dark basal keratinocytes compared to acetone. (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991).

Executive summary:

The study was conducted to determine the short -term effects of BzPo and compared them with the short-term effects of seven other organic peroxides. In the present paper, the authors evaluated short-term markers of tumor promotion ( hyperplasia, induction of dark basal keratinocytes and induction of ornithine decarboxylase activity) in order to correlate these parameters with the characteristics of the different compounds and to establish conditions for future tumor promotion experiments.

The authors concluded that, "The data presented here further support the association between free radicals and tumor promotion since all of the compounds, with the exception of one, DTBP, were active in inducing the short-term markers of tumor promotion." (Gimenez-Conti et al Carcinogenesis vol. 2 no.4 pp.563-569, 1991)

Description of key information

A number of investigations have been conducted over the years to determine the initiation/promotion activity of di-tert-butyl peroxide (DTBP).  Taken together, DTBP was inactive as an initiator or promotor. 

Additional information

DTBP did not induce short term markers of tumor promotion in two studies with mice. DTBP was inactive in promoting papillomas or carcinomas in initiated skin. No skin tumors developed following dermal application of DTBP for 81 weeks or for 80 weeks when applied prior to a promotor.

DTBP does not appear to have initiating or promoting activity.