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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented mechanistic investigation which meets basic scientific principles

Data source

Reference
Reference Type:
publication
Title:
Responses of Transgenic Mouse Lines p53+/- and Tg.AC to Agents Tested in Conventional Carcinogenicity Bioassays
Author:
Spalding, J.W. et al
Year:
2000
Bibliographic source:
Toxicological Sciences 53, 213-223

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
TEA was administered topically to homozygous female Tg¿AC mice.

The application site (from the dorsal interscapular region to a point ~1 cm from the base of the tail) was closely shaven prior to the first treatment; care was taken to avoid abrasions or cuts. The site of application (SOA) was shaved weekly or as needed prior to subsequent treatments. The topically applied TEA was solubilized in acetone and administered in 200-µl volumes 5x/week for 20 weeks.
Concurrent negative control groups were treated with 200 µl acetone. The positive control agent was TPA 1.25 µg 3x/week or 1.5 µg, 2x/week for 20 weeks. Dose groups comprised 13¿20 mice.
GLP compliance:
not specified
Type of method:
in vivo
Endpoint addressed:
carcinogenicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
TEA was from the same chemical batch (lot number) as that used in the conventional 2-year bioassay conducted by the National Toxicology Program.

Test animals

Species:
mouse
Strain:
other: Tg.AC
Sex:
female
Details on test animals and environmental conditions:
Homozygous female Tg¿AC mice (zetaglobin promoted v-Ha-ras on a FVB background) were obtained from the NTP breeding colony at Taconic Laboratory Animals and Services, (Germantown, NY) at 4 to 5 weeks.

The female Tg¿AC mice were group housed (either 4 or 5/cage) in polycarbonate shoebox style cages with absorbent hardwood bedding (Beta Chips, Northeastern Products Corp., Warrensburg, NY) in an AALAC-accredited facility. Female mice were group housed as a cost-saving measure. For future studies, single housing is recommended. The cages, with stainless steel tops, were changed at least twice a week. Mice were maintained on a 12-h (fluorescent) light:dark cycle. Relative humidity and temperature ranges across all studies were 7¿94% and 59¿82°F, respectively. At the start of the study, the Tg¿AC mice were 14 weeks of age. The Tg¿AC mice were held longer than usual before the study start, because animal availability and delivery occurred prior to the completion of the chemical characterization of the chemicals to be administered.

Each mouse was uniquely identified by tail tattoo prior to being placed on study. At study start, the mice were weighed to the nearest 0.1 g and were allocated to treatment or control groups using a stratified (by cage mean body weight for Tg¿AC mice) randomization procedure. Tg¿AC mice that exhibited evidence of skin abrasions or bite wounds at the site of chemical application were eliminated from the study. All mice had free access to tap water. Tg¿AC mice were maintained ad libitum on Purina Picolab No. 5058 chow (Granville Milling, Creedmoor, NC). We have routinely used the Picolab No. 5058 diet because, in our experience, Tg¿AC mice have a more favorable weight gain and are less aggressive. Picolab diet No.5058 has a 9% fat content in contrast to the NIH-07 formula, which has a 5% fat content.

Administration / exposure

Route of administration:
dermal
Vehicle:
acetone
Details on exposure:
TEA was administered topically to homozygous female Tg¿AC mice. The doses originally selected for the PCP study in Tg¿AC mice were 3.0, 6.0, or 12.0 mg/mouse. However, overt toxicity was observed at the two higher doses after the first application. Thereafter, for the remainder of the study, the dose group receiving 12.0 mg was placed on 0.75 mg/day, and the dose group receiving 6.0 mg was placed on 1.5 mg/day.

The application site (from the dorsal interscapular region to a point ~1 cm from the base of the tail) was closely shaven prior to the first treatment; care was taken to avoid abrasions or cuts. The site of application (SOA) was shaved weekly or as needed prior to subsequent treatments. The topically applied TEA was solubilized in acetone and administered in 200-µl volumes 5x/week for 20 weeks. At the time these studies were conducted, a 20-week dermal exposure period was the standard protocol used for studies in Tg¿AC mice. We now recommend a 26-week exposure period by all routes of administration. Concurrent negative control groups were treated with 200 µl acetone. The positive control agent was TPA 1.25 µg 3x/week or 1.5 µg, 2x/week for 20 weeks. Dose groups comprised 13¿20 mice.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose preparations were prepared approximately every 3 weeks and were stored at 4°C. The stability of these preparations under these conditions had been demonstrated previously by NTP. The preparations for each substance were analytically analyzed for accuracy three or four times during each study. With few exceptions, all dose formulation samples were within 10% of the targeted concentrations. Those several formulations that varied more than 10% were not considered to have a negative impact on the studies.
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
5x/week
Post exposure period:
no data
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 3, 10, 30 mg/animal
Basis:

Remarks:
Doses / Concentrations:
120, 400, 1200 mg/kg bw
Basis:

No. of animals per sex per dose:
13-20
Control animals:
yes, concurrent vehicle

Examinations

Examinations:
Clinical observations
Necropsy and histopathology
Positive control:
The positive control agent was TPA 1.25 µg 3x/week or 1.5 µg, 2x/week for 20 weeks.

Results and discussion

Details on results:
TEA was inactive in Tg¿AC mice. The average incidence of mice bearing papillomas was uniformly high among all groups of the TEA-treated mice, as well as in the acetone control group. However, the average tumor multiplicity was less than 1.0 in every instance. There was no indication that this low multiplicity was related to treatment, as the average multiplicity of papillomas for Tg¿AC mice was in the range observed for negative (vehicle) control groups (Spalding et al., 1999).

Any other information on results incl. tables

The haplo-insufficient p53 knockout (p53+/-) and zetaglobin v-Ha-ras (Tg¿AC) transgenic mouse models were compared to the conventional two rodent species carcinogen bioassay by prospectively testing nine chemicals. Seven of the chemicals classified as carcinogens in the conventional bioassay induced tumors in the liver or kidneys of B6C3F1mice, and one (pentachlorophenol) also induced tumors in other tissues. Only three chemicals, furfuryl alcohol, pyridine, and pentachlorophenol, induced tumors in rats. The tumorigenic effect of pyridine was seen in F344 rats but not in Wistar strain rats. None of the chemicals induced tumors in the p53 +/-transgenic mice, which is consistent with the absence of genotoxicity of these chemicals. Only two of the seven nongenotoxic carcinogens were positive in the Tg¿AC model (lauric acid diethanolamine and pentachlorophenol). These results show that these transgenic models do not respond to many chemicals that show strain- or species-specific responses in conventional bioassays.

TABLE: Prospective Evaluation of Chemical Activity in the Tg.AC Transgenic Mouse Model

Treatment

number of animals

incidence (%)

mean weeks to first tumor

multiplicity (tumors/total animals per group)

mean weeks to maximum tumor burden

survival at 20 weeks (%)

3.0 mg

14

4/14 (28.6)

11.8

5/14 (0.36)

13.8

11/14 (78.6)

10.0 mg

13

5/13 (38.5)

13.4

10/13 (0.77)

14.8

12/13 (92.3)

30.0 mg

19

4/19 (21.1)

8.8

10/19 (0.53)

13.5

15/19 (78.9)

Acetone (200 ul)

14

4/14 (28.6)

13.3

4/14 (0.29)

13.3

14/14 (100)

TPA (1.25 ug (2x/week))

20

19/20 (95.0)

7.6

390/20

12.4

9/20 (45.0)

Applicant's summary and conclusion