Registration Dossier

Toxicological information

Carcinogenicity

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Administrative data

Description of key information

Weight of evidence. A 27-week carcinogenicity study was performed with the test item in female p53 haploinsufficient mice, NTP protocol (GLP study). No evidence of carcinogenic activity was found. A 20-week carcinogenicity study was also performed in female Tg.AC hemizygous mice, NTP protocol (GLP study). The test item gave a positive result in this study, but this result was not conclusive.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 5, 1999 - July 9, 1999.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to other assay used for intermediate effect derivation
Principles of method if other than guideline:
Groups of 15 female p53 Haploinsufficient mice were dermally administered the test item at 6 concentrations for up to 27 weeks, and then necropsied and their organs examined histopathologically, in order to observe evidences of carcinogenic activity. NTP protocol.
GLP compliance:
yes
Species:
mouse
Strain:
other: B6.129-Trp53tmlBrd (N5)
Remarks:
p53 Haploinsufficient Mouse.
Details on species / strain selection:
The p53 haploinsufficient mouse model has been determined to be particularly useful for in vivo testing of mutagenic carcinogens [Donehower (1992); French (2001a,b), see 'Background material']: The heterozygous Trp53 null allele C57BL/6 (N5) mouse is susceptible to the rapid development of neoplasia by mutagenic carcinogens relative to control strains. This mouse model of chemical carcinogenesis demonstrates 1) dose-related rapid induction of tumors (26 wks), 2) multiple sites of carcinogen-specific tissue susceptibility, and 3) carcinogen-induced loss of heterozygosity involving the Trp53 wild-type allele or a p53 haploinsuficiency permitting mutation of other critical protooncogenes and/or inactivation of tumor suppressor genes driving tumorigenesis. Demonstration of mutation or loss of heterozygosity involving the Trp53 locus is consistent with a common finding in human cancers and supports extrapolation between rodents and humans.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Housing: individually housed
- Diet: Irradiated NTP-2000 formula pelleted diet (Ziegler Brothers Inc., Gardners, PA), ad libitum.
- Water: ad libitum tap water (Washington Suburban Sanitary Comission Potomac Plant) via automatic watering system.
- Acclimation period: 2 weeks.
- Before the study began, 5 mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from 5 randomly selected vehicle control mice. The sera were analyzed for antibody titers to rodent viruses; all results were negative.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 15%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12h/day

IN-LIFE DATES: From: January 5, 1999 (first dose) To: July 7-8, 1999
Route of administration:
dermal
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: treatment was applied to center of a shaved dorsal area posterior of the scapulae to the base of the tail.
- % coverage: < 10%

TEST MATERIAL
- Dose formulations were prepared every 3 weeks by mixing the test item and anhydrous ethanol to give the required concentration. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 35 days at room temperature, until April 1, 1999. Subsequent storage was performed at -20ºC.
- Concentration (if solution): 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw.
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item was moisture sensitive.
- Amount(s) applied (volume or weight with unit): 2 ml/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies of 0.38, 2 and 7 mg/L dose formulations of lot 00920TZ were conducted by the study laboratory by GC. Stability was confirmed for up to 35 days at room temperature, in sealed containers under a nitrogen headspace, and for up to 3 hours when exposed to light and air at room temperature.
Duration of treatment / exposure:
27 weeks.
Frequency of treatment:
5 days per week.
Dose / conc.:
0.75 mg/kg bw/day (nominal)
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Remarks:
Discontinued after 11 days due to the severity of skin lesions observed.
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
Discontinued after 8 days due to the severity of skin lesions observed.
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the doses for the study were based on the results of a 13-week toxicity study performed in B6C3F1 mice (see 'Cross-reference').
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded initially, weekly and at the end of the study.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded initially, weekly and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were performed on all animals.

HISTOPATHOLOGY: Yes
- Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.
- Complete histopathologic examinations were performed on all animals.
- Tissues examined: in addition to gross lesions and tissue masses, adrenal gland, bone with marrow, kidney, liver, lung, lymph nodes (mandibular, mediastinal, and mesenteric), mammary gland, ovary, parathyroid gland, pituitary gland, skin, spleen, stomach (forestomach and glandular), thymus, thyroid gland, trachea, and uterus.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
Chemical-related gross lesions of the skin at the site of application included reddening, red scars, red crusts, and ulcers. At the site of application, the incidences of focal chronic active inflammation of the dermis were increased in the 3 and 12 mg/kg groups, and the incidences of focal ulcer and focal chronic active inflammation of the subcutaneous tissue were increased in the 12 mg/kg group. In comparison, incidences of focal dermal chronic active inflammation, focal subcutaneous chronic active inflammation, focal epidermal hyperplasia, focal ulceration and focal sebaceous gland hyperplasia in the control site were less than those seen at the site of application and were less severe.
Mortality:
mortality observed, treatment-related
Description (incidence):
12 animals died or were sacrificed moribund prior to the end of the study: 3 from the 3 mg/kg group, 1 from the 6 mg/kg group, 8 from the 12 mg/kg group.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights of dosed groups were similar to those of the vehicle controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No neoplasms were attributed to the administration of the test item. At the site of application, the incidences of focal epidermal hyperplasia were significantly increased in the 1.5, 3, and 12 mg/kg groups.
Myeloid cell hyperplasia of the bone marrow occurred in the 3 mg/kg or greater groups, and the incidence of this lesion was significantly increased in the 12 mg/kg group. Increased hematopoiesis can occur with inflammatory conditions due to the physiological need for more white blood cells. Therefore, the increased incidence of myeloid cell hyperplasia is considered a relative change in response to the skin lesions at the site of application.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
dermal irritation
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes

Table 1. Survival and Body Weights of Female p53 Mice.

Dose

(mg/kg)

Survival (a)

Mean Body Weight (b)

Final Weight

Relative to Controls

(%)

Initial

Final

Change

0

15/15

18.6 ± 0.2

33.8 ± 1.2

15.3 ± 1.1

 

0.75

15/15

18.2 ± 0.3

32.1 ± 1.1

13.9 ± 1.1

95

1.5

15/15

18.3 ± 0.3

35.5 ± 1.2

17.2 ± 1.1

105

3

12/15 (c)

18.5 ± 0.3

32.3 ± 1.3

13.8 ± 1.2

96

6 (d)

14/15 (e)

18.0 ± 0.3

32.0 ± 1.4

14.0 ± 1.2

95

12 (f)

7/15 (g)

18.1 ± 0.3

33.6 ± 2.4

15.5 ± 2.2

99

(a) Number of animals surviving at 27 weeks / number in group. (b) Weights and weight changes are given as mean ± standard error. Subsequent calculations are based on animals surviving to the end of the study. Differences from the vehicle control are not significant by Dunnett's test. (c) Week of death: 7, 10, 24. (d) Treatment was discontinued after 11 days due to the severity of skin lesions at the site of application. (e) Week of death: 5. (f) Treatment was discontinued after 8 days due to the severity of skin lesions at the site of application. (g) Week of death: 8, 9, 9, 9, 9, 9, 9, 10.

Table 2. Incidences of Nonneoplastic Lesions of the Skin (Site of Application).

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg (a)

12

mg/kg (b)

Number Microscopically Examined

15

15

15

15

15

15

Dermis, Inflammation,

Chronic Active, Focal (c)

1 (1.0) (d)

0

0

13** (1.3)

2 (1.0)

9 ** (3.3)

Epidermis, Hyperplasia, Focal

0

1 (1.0)

5* (1.0)

11** (1.5)

1 (2.0)

8** (3.0)

Sebaceous Gland,

Hyperplasia, Focal

0

0

0

3 (1.7)

0

3 (2.0)

Ulcer, focal

0

0

0

2 (4.0)

1 (4.0)

8** (3.9)

Subcutaneous Tissue, Inflammation,

Chronic Active, Focal

0

0

0

2 (3.0)

0

8 ** (3.6)

* Significantly different (P0.05) from the vehicle control group by the Fisher exact test. ** P0.01. (a) Treatment was discontinued after 11 days because of the severity of skin lesions at the site of application. (b) Treatment was discontinued after 8 days because of the severity of skin lesions at the site of application. (d) Number of animals with lesion; Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

Table 2. Summary of the Incidence of Neoplasms in Female p53 Haploinsufficient Mice.

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

Disposition Summary

Animals initially in study

15

15

15

15

15

15

Early deaths

·        Natural deaths

 

 

 

1

1

1

·        Moribund

 

 

 

2

 

7

Survivors

 

 

 

 

 

 

·        Terminal sacrifice

15

15

15

12

14

7

Animals examined microscopically

15

15

15

15

15

15

Alimentary System

Mesentery

 

 

 

(1)

 

 

·        Lymphoma malignant

 

 

 

1 (100%)

 

 

Hematopoietic System

Lymph node, mandibular

(15)

(15)

(14)

(14)

(14)

(14)

·        Histocytic sarcoma

 

 

 

 

1 (7%)

 

Lymph node, mesenteric

(14)

(15)

(15)

(15)

(14)

(14)

·        Histocytic sarcoma

 

 

 

 

1 (7%)

 

Lymph node, mediastinal

(14)

(15)

(15)

(12)

(13)

(13)

·        Histocytic sarcoma

 

 

 

 

1 (8%)

 

·        Lymphoma malignant

 

 

 

1 (8%)

 

 

Thymus

(15)

(15)

(15)

(15)

(14)

(13)

·        Histocytic sarcoma

 

 

 

 

1 (7%)

 

·        Lymphoma malignant

 

 

 

1 (7%)

 

 

Respiratory System

Lung

(15)

(15)

(15)

(15)

(14)

(15)

·        Lymphoma malignant

 

 

 

1 (7%)

 

 

Systemic Lesions

Multiple organs (b)

(15)

(15)

(15)

(15)

(15)

(15)

·        Histiocytic sarcoma

 

 

 

 

1 (7%)

 

·        Lymphoma malignant

 

 

 

1 (7%)

 

 

Systems Examined with No Neoplasms Observed:

Cardiovascular System

Endocrine System

General Body System

Genital System

Intergumentary System

Musculoskeletal System

Nervous System

Special Senses System

Urinary System

Neoplasm Summary

Total animals with primary neoplasms (c)

 

 

 

1

1

 

Total primary neoplasms

 

 

 

1

1

 

Total animals with malignant neoplasms

 

 

 

1

1

 

Total malignant neoplasms

 

 

 

1

1

 

Table 3. Summary of the Incidence of Nonneoplastic Lesions in Female p53 Haploinsufficient Mice.

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

Disposition Summary

Animals initially in study

15

15

15

15

15

15

Early deaths

·         Natural deaths

 

 

 

1

1

1

·         Moribund

 

 

 

2

 

7

Survivors

 

 

 

 

 

 

·         Terminal sacrifice

15

15

15

12

14

7

Animals examined microscopically

15

15

15

15

15

15

Alimentary System

Liver

(15)

(15)

(15)

(15)

(14)

(15)

·         Hematopoietic cell proliferation

 

 

 

 

 

1 (7%)

·         Hematopoietic cell proliferation,

focal

 

 

 

 

 

1 (7%)

·         Infiltration cellular, focal, lymphocyte

 

1 (7%)

 

4 (27%)

1 (7%)

2 (13%)

·         Inflammation, chronic active, focal

 

 

 

 

 

1 (7%)

·         Hepatocyte, necrosis, focal

4 (27%)

6 (40%)

5 (33%)

4 (27%)

2 (14%)

1 (7%)

·         Periportal, vacuolization cytoplasmic,

focal

 

 

 

1 (7%)

 

 

Stomach, glandular

(15)

(15)

(15)

(15)

(14)

(14)

·         Infiltration cellular, focal, lymphocyte

 

 

 

1 (7%)

 

 

Cardiovascular System: none

Endocrine System

Adrenal cortex

(15)

(15)

(15)

(15)

(14)

(13)

·         Subcapsular, hyperplasia

 

 

 

 

 

1 (8%)

·         Subcapsular, hyperplasia, focal

13 (89%)

13 (87%)

12 (80%)

13 (87%)

14 (100%)

9 (69%)

Thyroid gland

(13)

(15)

(15)

 

(13)

(13)

·         Ectopic thymus

 

2 (13%)

3 (20%)

1 (7%)

1 (8%)

 

·         Inflammation, chronic active, focal

1 (8%)

 

 

 

 

 

General Body System: none

Genital System

Ovary

(15)

(15)

(15)

(15)

(14)

(14)

·         Atrophy

 

 

1 (7%)

 

 

1 (7%)

Uterus

(15)

(15)

(15)

(15)

(14)

(14)

·         Endometrium, hyperplasia, cystic

14 (93%)

15 (100%)

15 (100%)

13 (87%)

13 (93%)

7 (50%)

Hematopoietic System

Bone marrow

(15)

(15)

(15)

(15)

(14)

(15)

·         Myeloid cell, hyperplasia

 

 

 

3 (20%)

1 (7%)

8 (53%)

Lymph node

 

 

 

(1)

 

 

·         Inguinal, hyperplasia, lymphoid

 

 

 

1 (100%)

 

 

Lymph node, mandibular

(15)

(15)

(14)

(14)

(14)

(14)

·         Hyperplasia, lymphoid

1 (7%)

 

 

2 (14%)

3 (21%)

 

Lymph node, mediastinal

(14)

(15)

(15)

(12)

(13)

(13)

·         Hyperplasia, lymphoid

 

 

1 (7%)

 

 

 

Spleen

(15)

(15)

(15)

(15)

(14)

(14)

·         Hematopoietic cell proliferation

15 (100%)

15 (100%)

15 (100%)

15 (100%)

13 (93%)

13 (93%)

·         Pigmentation

 

 

 

 

 

1 (7%)

Thymus

(15)

(15)

(15)

(15)

(14)

(13)

·         Atrophy, diffuse

 

 

 

 

 

2 (15%)

·         Atrophy, focal

 

2 (13%)

1 (7%)

 

 

 

Intergumentary system

Mammary gland

(15)

(15)

(15)

(15)

(14)

(14)

·         Inflammation, chronic active, focal

 

 

 

 

 

1 (7%)

Skin

(15)

(15)

(15)

(15)

(15)

(15)

·         Control, ulcer, focal

 

 

 

1 (7%)

 

2 (13%)

·         Dermis, epidermis, hyperplasia, focal

 

 

 

 

 

1 (7%)

·         Dermis, control, inflammation,

chronic active, focal

 

 

 

3 (20%)

1 (7%)

2 (13%)

·         Dermis, skin, site of application, fibrosis

 

 

 

 

 

1 (7%)

·         Dermis, skin, site of application,

inflammation, chronic active, focal

1 (7%)

 

 

13 (87%)

2 (13%)

9 (60%)

·         Epidermis, control, hyperplasia, focal

 

 

 

2 (13%)

 

1 (7%)

·         Epidermis, skin, site of application,

hyperplasia, diffuse

 

 

1 (7%)

2 (13%)

 

 

·         Epidermis, skin, site of application,

hyperplasia, focal

 

1 (7%)

5 (33%)

11 (73%)

 

8 (53%)

·         Sebaceous gland, control, hyperplasia, focal

 

 

 

1 (7%)

 

1 (7%)

·         Sebaceous gland, skin, site of application,

hyperplasia, focal

 

 

 

3 (20%)

 

3 (20%)

·         Skin, site of application, hyperkeratosis,

diffuse

 

 

 

1 (7%)

1 (7%)

 

·         Skin, site of application, parakeratosis, focal

 

 

 

1 (7%)

1 (7%)

 

·         Skin, site of application, ulcer, focal

 

 

 

2 (13%)

1 (7%)

8 (53%)

·         Subcutaneous tissue, control, inflammation, chronic active, focal

 

 

 

1 (7%)

 

2 (13%)

·         Subcutaneous tissue, skin, site of application,

inflammation, chronic, focal

 

 

 

 

1 (7%)

 

·         Subcutaneous tissue, skin, site of application,

inflammation, chronic active, focal

 

 

 

2 (13%)

 

8 (53%)

Musculoskeletal System: none

Nervous System: none

Respiratory System

Lung

(15)

(15)

(15)

(15)

(14)

(15)

·         Alveolar epithelium, hyperplasia, focal

 

 

1 (7%)

 

 

 

·         Alveolar epithelium, inflammation, chronic active, focal

 

1 (7%)

 

 

 

 

·         Alveolus, inflammation, chronic active, focal

1 (7%)

 

 

 

1 (7%)

1 (7%)

·         Perivascular, infiltration cellular, focal, lymphocyte

1 (7%)

 

 

 

2 (14%)

 

Special Senses System: none

Urinary System:

Kidney

(15)

(15)

(15)

(15)

(14)

(14)

·         Infiltration cellular, focal, lymphocyte

 

1 (7%)

 

 

 

 

·         Pelvis, infiltration cellular, focal, lymphocyte

 

 

 

1 (7%)

 

 

(a) Number of animals examined microscopically at the site and the number of animals with lesion.

Conclusions:
Under test conditions, there was no evidence of carcinogenic activity of the test item.
Executive summary:

A 27-week carcinogenicity study was performed with the test item in female p53 haploinsufficient mice (GLP study). Groups of 15 female mice were administered 0, 0.75, 1.5, 3. 6 or 12 mg/kg bw of test item in ethanol, 5 days per week for 27 weeks. Dosing of the 6 and 12 mg/kg groups was discontinued after 11 and 8 days due to the severity of skin lesions at the site of application. Twelve animals died or were sacrificed moribund prior to the end of the study: 8 from the 12 mg/kg group, 1 from the 6 mg/kg group, and 3 from the 3 mg/kg group. No neoplasms were attributed to the test item. Under the conditions of this 27 -week dermal study, there was no evidence of carcinogenic activity of the test item.

Endpoint:
carcinogenicity: dermal
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 15, 1995 - November 2, 1995.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to other assay used for intermediate effect derivation
Principles of method if other than guideline:
Groups of 10 female Tg.AC hemizygous mice were dermally administered the test item at 6 concentrations for up to 20 weeks, and then necropsied and their organs examined histopathologically, in order to observe evidences of carcinogenic activity. NTP protocol.
GLP compliance:
yes
Species:
mouse
Strain:
FVB
Remarks:
FVB/N-TgN(v-Ha-ras)Led (Tg.AC) hemizygous mice.
Details on species / strain selection:
The Tg.AC hemizygous mouse model responds to both genoxotic and nongenotoxic carcinogens [Spalding (1999, 2000); Tennant (2001), see 'Background information']: The Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermall papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter of phenotype that defines the activity of the test article. The Tg.AC (v-Ha-ras) transgenic mouse model provides a reporter phenotype of skin papillomas in response to either genotoxic or nongenotoxic carcinogens.
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services, Inc. (Germantown, NY).
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks.
- Housing: individually housed
- Diet: ad libitum NIH-07 open formula pelleted diet (Ziegler Brothers Inc., Gardners, PA), changed weekly.
- Water: ad libitum tap water (Washington Suburban Sanitary Comission Potomac Plant) via automatic watering system.
- Acclimation period: 2 weeks.
- Before the study began, 5 mice were randomly selected for parasite evaluation and gross observation for evidence of disease. Blood was collected from 5 randomly selected vehicle control mice at study termination and from one mouse that was sacrificed moribund during the study. The sera were analyzed for antibody titers to rodent viruses; all results were negative.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 50 ± 15%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12h/day
Route of administration:
dermal
Vehicle:
ethanol
Details on exposure:
TEST SITE
- Area of exposure: treatment was applied to center of a shaved dorsal area posterior of the scapulae to the base of the tail.
- % coverage: < 10%

TEST MATERIAL
- Dose formulations were prepared every 3 weeks by mixing the test item and anhydrous ethanol to give the required concentration. The dose formulations were stored in sealed vials under a headspace of inert gas for up to 35 days at room temperature.
- Concentration (if solution): 0, 0.75, 1.5, 3, 6, or 12 mg/kg bw.
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test item was moisture sensitive.
- Amount(s) applied (volume or weight with unit): 2 ml/kg bw.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability studies of 0.38, 2 and 7 mg/L dose formulations of lot 00920TZ were conducted by the study laboratory by GC. Stability was confirmed for up to 35 days at room temperature, in sealed containers under a nitrogen headspace, and for up to 3 hours when exposed to light and air at room temperature.
Duration of treatment / exposure:
20 weeks.
Frequency of treatment:
5 days per week.
Dose / conc.:
0.75 mg/kg bw/day (nominal)
Dose / conc.:
1.5 mg/kg bw/day (nominal)
Dose / conc.:
3 mg/kg bw/day (nominal)
Dose / conc.:
6 mg/kg bw/day (nominal)
Dose / conc.:
12 mg/kg bw/day (nominal)
Remarks:
Discontinued after 8 applications due to the severity of skin lesions observed.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the doses for the study were based on the results of 13-week toxicity studies performed in B6C3F1 mice (see 'Cross-reference').
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical findings were recorded initially, weekly and at the end of the study.

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations:

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were recorded initially, weekly and at the end of the study.

OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsies were performed on all animals. The heart, right kidney, liver, lung and thymus were weighed.

HISTOPATHOLOGY: Yes
- Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin.
- Complete histopathologic examinations were performed on all vehicle control mice, all 12 mg/kg mice, and all mice that died before the end of the study. In addition, the skin was examined in all remaining dosed groups.
- Tissues examined: all gross lesions and tissue masses, plus adrenal gland, bone with marrow, brain, clitoral gland, esophagus, gallbladder, heart with aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, salivary gland, skin, spleen, stomach (forestomach and glandular), thymus, thyroid gland, trachea, urinary bladder and uterus.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical findings consisted of dose-related skin irritation at the site of the application in groups treated with 1.5 mg/kg or greater.
Dermal irritation (if dermal study):
effects observed, treatment-related
Description (incidence and severity):
Dose-related skin irritation at the site of the application in groups treated with 1.5 mg/kg or greater.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
13 animals died or were sacrificed moribund prior to the end of the study: 3 each from the vehicle control and 0.75 mg/kg groups, 4 from the 3 mg/kg group, 2 from the 6 mg/kg group, and 1 from the 12 mg/kg group. Overall, the survival was within the range known for the Tg.AC hemizygous mouse.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights of dosed groups of mice were similar to those of the vehicle controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative weights of the liver in 1.5 mg/kg or greater mice were significantly greater than those in the vehicle controls.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the site of application, the incidences of chronic active inflammation of the dermis and epidermal hyperplasia were significantly increased in the 3 and 6 mg/kg groups.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At the site of application, masses and/or nodules (papillomas) were observed: 0/10, 0/10, 1/10, 4/10, 6/10, 9/10 for the doses 0, 0.75, 1.5, 3, 6, 12, respectively. The incidences of squamous cell papilloma and multiplicity of squamous cell papiloma were increased in a dose-related manner. One squaomus cell carcinoma occured at the site of appication in a 6 mg/kg mouse.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
histopathology: neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 mg/kg bw/day (nominal)
System:
integumentary
Organ:
skin
Treatment related:
yes
Dose response relationship:
yes

Table 1. Survival and Body Weights of Females Tg.Ac Hemizygous Mice.

Dose

(mg/kg)

Survival (a)

Mean Body Weight (b)

Final Weight

Relative to Controls

(%)

Initial

Final

Change

0

7/10 (c)

20.6 ± 0.4

26.8 ± 1.5

6.0 ± 1.2

 

0.75

7/10 (d)

20.9 ± 0.3

27.7 ± 1.0

7.1 ± 1.0

103

1.5

10/10

21.0 ± 0.4

26.7 ± 0.7

5.7 ± 0.7

100

3

6/10 (e)

20.8 ± 0.3

27.5 ± 0.8

6.3 ± 0.8

103

6

8/10 (f)

20.5 ± 0.4

28.8 ± 0.9

8.3 ± 0.7

108

12 (g)

9/10 (h)

20.5 ± 0.4

27.4 ± 0.9

27.4 ± 0.9

102

(a) Number of animals surviving at 20 weeks/number initially in group. (b) Weights and weight changes are given as mean ± standard error. Subsequent calulations are based on animals surviving to the end of the study. Differences from the vehicle control group are not significant by Dunnett's test. (c) Week of death: 9, 12, 15. (d) Week of death: 7, 12, 19. (e) Week of death: 8, 12, 14, 17. (f) Week of death: 11, 18. (g) Treatment was discontinued after 8 days because of the severity of skin lesions at the site of application. (h) Week of death: 11.

Table 2. Incidences of Neoplasms and Nonneoplastic Lesions of the Skin (Site of Application) in Female Tg.AC Hemizygous Mice.

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg (a)

No. Examined Macroscopically

10

10

10

10

10

10

Dermis, Inflammation, Chronic Active (b)

0

0

3 (1.7) (c)

5* (1.8)

8** (2.0)

0

Epidermis, Hyperplasia

0

0

0

6** (1.8)

7** (2.0)

1 (2.0)

Squamous Cell Papilloma,

Multiple

0

0

1

0

3

5*

Squamous Cell Papilloma (includes multiple)

0

0

1

0

3

5*

Squamous Cell Carcinoma

0

0

0

0

1

0

*Significantly different (P ≤ 0.05) from the vehicle control group by the Fisher exact test. **P≤ 0.01. (a) Treatment was discontinued after 8 days because of the severity of skin lesions at the site of application. (b) Number of animals with lesion. (c) Average severity grade of lesions in affected animals: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked.

Table 3. Summary of the Incidence of Neoplasms in Female Tg.AC Hemizygous Mice.

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

Disposition Summary

Animals initially in study

10

10

10

10

10

10

Early deaths

 

 

 

 

 

 

·        Accidental death

 

 

 

1

 

 

·        Natural deaths

1

1

 

1

 

1

·        Moribund

2

2

 

2

2

 

Survivors

 

 

 

 

 

 

·        Terminal sacrifice

7

7

10

6

8

9

Animals examined microscopically

10

10

10

10

10

10

Alimentary System

Liver

(10)

(6)

(6)

(7)

(10)

(10)

·        Leukemia erythrocytic

1 (10%)

 

2 (33%)

 

1 (10%)

 

Pancreas

(10)

(3)

 

(4)

(2)

(10)

·        Leukemia erythrocytic

 

 

 

 

1 (50%)

 

Stomach, forestomach

(10)

(4)

(2)

(5)

(4)

(10)

·        Squamous cell papilloma

 

2 (50%)

2 (100%)

1 (20%)

1 (25%)

 

Tooth

(4)

(5)

(6)

(4)

(5)

(5)

·        Odontoma

3 (75%)

5 (100%)

6 (100%)

3 (75%)

5 (100%)

5 (100%)

Endocrine System

Thyroid gland

(9)

(4)

 

(4)

(2)

(10)

·        C-cell, adenoma

 

1 (25%)

 

 

 

 

Hematopoietic System

Lymph node

 

(1)

 

 

(1)

 

·        Leukemia erythrocytic

 

 

 

 

1 (100%)

 

Spleen

(10)

(5)

(5)

(4)

(4)

(10)

·        Leukemia erythrocytic

 

 

2 (40%)

 

1 (25%)

 

Thymus

(9)

(3)

 

(3)

(2)

(10)

·        Lymphoma malignant

1 (11%)

 

 

 

 

 

Intergumentary System

Skin

(10)

(10)

(10)

(10)

(10)

(10)

Squamous cell papilloma

·        Skin, site of application,

·        Squamous cell carcinoma

 

 

1 (10%)

 

 

1 (10%)

·        Skin, site of application,

·        Squamous cell carcinoma

 

 

 

 

1 (10%)

 

·        Skin, site of application,

·        Squamous cell papilloma

 

 

 

3 (30%)

3 (30%)

3 (30%)

·        Skin, site of application,

·        Squamous cell papilloma, multiple

 

 

1 (10%)

 

3 (30%)

5 (50%)

Systemic Lesions

Multiple organs (b)

(10)

(10)

(10)

(10)

(10)

(10)

·        Lymphoma erythrocytic

1 (10%)

 

2 (20%)

 

1 (10%)

 

·        Lymphoma malignant

1 (10%)

 

 

 

 

 

Systems Examined with No Neoplasms Observed:

Cardiovascular System

General Body System

Genital System

Musculoskeletal System

Nervous System

Respiratory System

Special Senses System

Urinary System

Neoplasm Summary

Total animals with primary neoplasms (c)

5

6

9

6

8

10

Total primary neoplasms

5

8

12

7

14

14

Total animals with benign neoplasms

3

6

8

6

8

10

Total benign neoplasms

3

8

10

7

12

14

Total animals with malignant neoplasms

2

 

2

 

2

 

Total malignant neoplasms

2

 

2

 

2

 

Table 4. Summary of Nonneoplastic Lesions in Female Tg.AC Hemizygous Mice. 

 

Vehicle

control

0.75

mg/kg

1.5

mg/kg

3

mg/kg

6

mg/kg

12

mg/kg

Disposition Summary

Animals initially in study

10

10

10

10

10

10

Early deaths

 

·        Accidental death

 

 

 

1

 

 

·        Natural deaths

1

1

 

1

 

1

·        Moribund

2

2

 

2

2

 

Survivors

 

·        Terminal sacrifice

7

7

10

6

8

9

Animals examined microscopically

10

10

10

10

10

10

Alimentary system

Gallbladder

(9)

(3)

 

(3)

(2)

(7)

·        Inflammation

 

1 (33%)

 

 

 

1 (14%)

Intestine large, rectum

(9)

(3)

 

(4)

(2)

(9)

·        Anus, inflammation

 

 

 

 

1 (50%)

 

Liver

(10)

(6)

(6)

(7)

(10)

(10)

·        Fibrosis

 

1 (17%)

 

 

 

1 (10%)

·        Hematopoietic cell proliferation

1 (10%)

1 (17%)

 

 

2 (20%)

1 (10%)

·        Inflammation, chronic active,

·        focal

 

3 (50%)

3 (50%)

4 (57%)

6 (60%)

2 (20%)

·        Inflammation, focal

3 (30%)

 

 

 

 

5 (50%)

·        Necrosis

 

1 (17%)

1 (17%)

2 (29%)

1 (10%)

 

·        Necrosis, focal

1 (10%)

 

 

 

 

1 (10%)

Stomach, forestomach

(10)

(4)

(2)

(5)

(4)

(10)

·        Inflammation

 

1 (25%)

 

 

 

 

Stomach, glandular

(10)

(3)

 

(4)

(2)

(10)

·        Inflammation

1 (10%)

 

 

 

 

1 (10%)

·        Serosa, fibrosis

 

 

 

 

 

1 (10%)

Tooth

(4)

(5)

(6)

(4)

(5)

(5)

·        Inflammation, chronic active

 

 

 

 

 

1 (20%)

Cardiovascular System

Heart

(10)

(3)

 

(4)

(2)

(10)

·        Inflammation, acute

 

 

 

 

 

1 (10%)

·        Mineralization, focal

 

 

 

 

 

1 (10%)

Endocrine system

Pituitary gland

(10)

(2)

 

(3)

(1)

(8)

·        Pars distalis, necrosis, focal

 

 

 

 

1 (10%)

 

General Body System: none

Genital System

Clitoral gland

(8)

(1)

 

(4)

(1)

(9)

·        Inflammation

 

 

 

 

 

1 (11%)

Uterus

(10)

(3)

 

(4)

(2)

(10)

·        Cyst

1 (10%)

 

 

 

 

1 (10%)

·        Bilateral, cyst

 

 

 

 

 

1 (10%)

Hematopoietic System

Bone marrow

(10)

(3)

 

(4)

(2)

(10)

·        Hyperplasia

3 (30%)

 

 

2 (50%)

1 (50%)

2 (20%)

·        Myeloid cell, hyperplasia

 

1 (33%)

 

 

 

 

Lymph node

 

(1)

 

 

(1)

 

·        Bronchial, infiltration cellular,

plasma cell

 

1 (100%)

 

 

 

 

Lymph node, mandibular

(10)

(3)

(1)

(4)

(3)

(10)

·        Hematopoietic cell proliferation

 

1 (33%)

 

 

 

 

·        Infiltration cellular, histiocyte

1 (11%)

 

 

 

 

 

·        Necrosis, focal

1 (11%)

 

 

1 (25%)

 

 

Spleen

(10)

(5)

(5)

(4)

(4)

(10)

·        Fibrosis

 

 

 

 

 

1 (10%)

·        Hematopoietic cell proliferation

8 (80%)

4 (80%)

4 (80%)

3 (75%)

3 (75%)

9 (90%)

·        Lymphoid follicle, depletion cellular

 

 

 

 

 

1 (10%)

Thymus

(9)

(3)

 

(3)

(2)

(10)

·        Atrophy

1 (11%)

2 (67%)

 

1 (33%)

2 (100%)

1 (10%)

·        Hemorrhage

 

 

 

 

 

1 (10%)

Intergumentary System

Skin

(10)

(10)

(10)

(10)

(10)

(10)

·        Hemorrhage

 

 

 

1 (10%)

 

 

·        Hyperplasia

 

 

 

 

 

1 (10%)

·        Inflammation, chronic active

 

 

1 (10%)

 

 

 

·        Dermis, inflammation

 

 

 

 

1 (10%)

 

·        Dermis, inflammation, chronic active

 

 

 

1 (10%)

 

 

·        Dermis, skin, site of application,

·        inflammation, chronic active

 

 

3 (30%)

5 (50%)

8 (80%)

 

·        Epidermis, skin, site of application,

hyperplasia

 

 

 

6 (60%)

7 (70%)

1 (10%)

Musculoskeletal System

Bone

(10)

(3)

 

(4)

(2)

(10)

·        Synovial tissue, inflammation

1 (10%)

 

 

 

 

 

Nervous System

Brain

(10)

(3)

 

(4)

(2)

(10)

·        Hemorrhage, acute

 

 

 

1 (25%)

 

 

Respiratory System

Larynx

 

 

 

 

 

(1)

·        Inflammation

 

 

 

 

 

1 (10%)

Lung

(10)

(3)

 

(4)

(2)

(10)

·        Hemorrhage, acute

 

 

 

1 (25%)

 

 

·        Inflammation, chronic active

 

 

 

 

 

4 (40%)

Nose

(10)

(3)

 

(4)

(5)

(10)

·        Inflammation

 

 

 

 

3 (60%)

1 (10%)

·        Respiratory epithelium, inflammation

 

 

 

 

 

1 (10%)

Special Senses System: none

Urinary System

Kidney

(10)

(3)

 

(4)

(2)

(10)

·        Inflammation, chronic active

 

1 (33%)

 

 

 

1 (10%)

·        Bilateral, inflammation

 

 

 

 

 

1 (10%)

·        Cortex, cyst

1 (10%)

 

 

 

 

 

(a)     Number of animals examined microscopically at the site and the number of animals with lesion.

Conclusions:
The test item gave a positive result in this study, due to a significant increase of squamous cell papilloma of the skin at the site of application. However, in the current study, it is not possible to determine how the test item induced papilloma with a dose response that was similar to that of irritation, inflammation, and hyperplasia at the site of application.
Executive summary:

A 20-week carcinogenicity study was performed with the test item in female Tg.AC hemizygous mice (GLP study). Groups of 10 female mice were administered 0, 0.75, 1.5, 3. 6 or 12 mg/kg bw of test item in ethanol, 5 days per week for up to 20 weeks. Due to the severity of skin lesions, treatment was discontinued in the 12 mg/kg group after 8 applications. Although 13 animals died prior to the end of the study, no deaths related to the test item were observed, as the survival rate was within the acceptable range for the species. Incidences of chronic active inflammation of the dermis and epidermal hyperplasia were significantly increased in the 3 and 6 mg/kg groups. At the site of application, the incidences of squamous cell papilloma were increased in a dose-related manner. The test item gave a positive result in this study. However, in the current study, it is not possible to determine how the test item induced papilloma with a dose response that was similar to that of irritation, inflammation, and hyperplasia at the site of application.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
mouse
Quality of whole database:
Two studies available, with a Klimisch score of 2.

Additional information

Weight of evidence. A 27-week carcinogenicity study was performed with the test item in female p53 haploinsufficient mice (GLP study). Groups of 15 female mice were administered 0, 0.75, 1.5, 3. 6 or 12 mg/kg bw of test item in ethanol, 5 days per week for 27 weeks. Dosing of the 6 and 12 mg/kg groups was discontinued after 11 and 8 days due to the severity of skin lesions at the site of application. No neoplasms were attributed to the test item. Under the conditions of this 27 -week dermal study, there was no evidence of carcinogenic activity of the test item. A 20-week carcinogenicity study was also performed, with the test item in female Tg.AC hemizygous mice (GLP study). Groups of 10 female mice were administered 0, 0.75, 1.5, 3. 6 or 12 mg/kg bw of test item in ethanol, 5 days per week for up to 20 weeks. Due to the severity of skin lesions, treatment was discontinued in the 12 mg/kg group after 8 applications. Although 13 animals died prior to the end of the study, no deaths related to the test item were observed, as the survival rate was within the acceptable range for the species. Incidences of chronic active inflammation of the dermis and epidermal hyperplasia were significantly increased in the 3 and 6 mg/kg groups. At the site of application, the incidences of squamous cell papilloma were increased in a dose-related manner. The test item gave a positive result in this study. However, in the current study, it is not possible to determine how the test item induced papilloma with a dose response that was similar to that of irritation, inflammation, and hyperplasia at the site of application.

Justification for classification or non-classification

Based on the available information, the substance is not classified for carcinogenicity according to CLP Regulation (EC) no. 1272/2008.