Registration Dossier

Administrative data

Endpoint:
specific investigations: other studies
Type of information:
experimental study
Adequacy of study:
supporting study
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.

Data source

Reference
Reference Type:
publication
Title:
Exposure of mouse skin to organic peroxides: subchronic effects related to carcinogenic potential
Author:
Hanausek, M.
Year:
2004
Bibliographic source:
Carcinogenesis vol.25 no.3 pp.431±437, 2004
Report Date:
2004

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
t-butyl peroxybenzoate (TBPB) (lot no. 603894/JAD-6-25, 98.0% pure)

Test animals

Species:
mouse
Strain:
Sencar
Sex:
female
Details on test animals 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)

Administration / exposure

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 week for 4, or 8, or 12 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
100 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.

8 and 12 week studies. TBPB was
also evaluated for its ability to induce increases in the above three parameters
when topically applied for 8 or 12 weeks at the maximal doses stated
above. Specimens of dosed skin were harvested 2 days after final dosing and
increases in the mentioned parameters were compared with those produced by
the vehicle or positive (DMBA or TPA) controls. Specimens of dosed skin
were also evaluated for the occurrence of mutations in codons 12, 13 and 61 of
the c-Ha-ras protooncogene. Mice dosed with MNNG, B[a]P, urethane or
DMBA were used as controls for mutations in c-Ha-ras at codons 12, 13 or
61, respectively (23±27,30). When
present, the incidence and multiplicity of skin tumors are reported.

16 week initiation/promotion protocol. The occurrence of mutations in the
c-Ha-ras protooncogene was also examined in mice treated with a single dose
of TBPB (100 mmol) followed by 12 weeks of
promotion by TPA (2 mg, 2/wk). Mice dosed with single topical applications
of MNNG, B[a]P, urethane or DMBA, followed by 12 weeks of TPA promotion,
were used as controls for mutations in c-Ha-ras at codon 12, 13 or 61,
respectively. Dosed skins, with tumors
when present, were harvested 4 weeks following termination of TPA promotion.
When present, the incidence and multiplicity of skin tumors are reported. ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004

Examinations

Examinations:
Cutaneous effects related to carcinogenic
potential, namely effects on sustained epidermal hyperplasia,
dermal inflammation, oxidative damage to skin DNA,
and mutation of codon 12, 13 or 61 of the c-Ha-ras protooncogene.
Positive control:
See details on study design

Results and discussion

Details on results:
( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004)

"Four week exposures were adequate to demonstrate the effects on sustained epidermal hyperplasia, dermal cellularity and oxidative damage to DNA:TBPB produced increases in sustained epidermal hyperplasia, dermal cellularity and oxidative damage to DNA.

Study using 8 and 12 week exposures to TBPB: Mutations in codon 61 were detected after 8 weeks exposure in five of five mice treated with
100 nmol DMBA (2/wk). No mutations in codons 12 and 13 were detected in DNA isolated from mice treated with B[a]P (50 or 200 mmol, 2/wk) or with the highest dose of MNNG (1 mmol, 2/wk). No mutations were noted in mice treated with the TBPB.

After 12 weeks treatment, mutations in codon 61 were detected in groups receiving the following treatments: 10 nmol DMBA (2/wk), one of five mice; 100 nmol DMBA, (1/wk), four of five mice; 100 nmol DMBA (2/wk) five of five mice; 670 mmol urethane (2/wk), one of five mice. No mutations in codon 61 were detected at the highest doses of TBPB. Mutation in codon 12 of the Haras oncogene was detected in one of five mice treated with 1 mmol MNNG (2/wk) and mutation in codon 13 was detected in three of five mice treated with 200 mmol B[a]P (2/wk). The codon 12 mutation detected in the MNNG group was weak, but nevertheless clearly visible. In the case of B[a]P, the bands indicating mutations at codon 13 were readily detected. None of the DNA from mice treated with the highest doses of TBPB (2/wk) exhibited mutations in codon 12 or 13 of the Ha-ras oncogene.

16 week initiation/promotion protocol using TBPB: No mutations of c-Ha-ras or tumors were detected in mice treated with a single
`initiating' dose of TBPB and promoted with TPA."

Applicant's summary and conclusion

Conclusions:
The authors conclude: "TBPB exhibited significant increases in all three biomarkers associated with tumor promoting activity. TBPB did not produced detectable mutations in the c-Ha-ras protooncogene, indicating it is not likely to possess tumor initiating or complete carcinogenic activity." ( 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 TBPB.

TBPB 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. TBPB exhibited significant increases in all three biomarkers associated with tumor promoting activity.

TBPB was further examined for its ability to produce mutations in codons 12, 13 and 61 of the c-Ha-ras protooncogene, i.e. those mutations known to be involved in the initiation of mouse skin tumors. Evaluations were performed using SENCAR mice dosed topically for 8 or 12 weeks in a complete carcinogenesis protocol or 16 weeks in an initiation/promotion protocol using 7,12- dimethylbenz[a]anthracene, urethane, benzo[a]pyrene and N-methyl-N0-nitro-N-nitrosoguanidine as positive controls. TBPB did not produced detectable mutations in the c-Ha-ras protooncogene, indicating it is not likely to possess tumor initiating or complete carcinogenic activity ( Hanausek, M. et al Carcinogenesis vol.25 no.3 pp.431±437, 2004).