1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to guideline; under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

EC Number:

603-837-5

Cas Number:

134605-64-4

Molecular formula:

C20H18ClF3N2O6

IUPAC Name:

1-(allyloxy)-2-methyl-1-oxopropan-2-yl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-3,6-dihydropyrimidin-1(2H)-yl]benzoate

Details on test material:

- Substance type: Tan powder
- Physical state: Solid
- Stability under test conditions: Stable under test conditions
- Storage condition of test material: Room temperature

Test animals

Species:

rat

Strain:

other: Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: Not reported
- Weight at study initiation: males 208.6 - 243.0 g; females 229.5 - 255.8 g
- Fasting period before study: Not reported
- Housing: Housed individually in Macrolon cages type 3 (area: 900 square centimeters) with wire mesh tops and granulated soft wood bedding
- Diet (e.g. ad libitum): certified standard pelleted diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Air changes (per hr): aproximately 15
- Photoperiod (hrs dark / hrs light): 12h light / 12h dark

IN-LIFE DATES: From: October 17, 1995 To: November 22, 1995

Administration / exposure

Type of coverage:

occlusive

Vehicle:

other: 0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.

Details on exposure:

TEST SITE
- Area of exposure: Dorsal area (The test article/vehicle suspension was applied to the right side of clipped area (=skin application site). The vehicle only was applied to the left side of clipped area (=skin remote site).
- % coverage: At least 10%
- Type of wrap if used: Gauze patches covered with aluminum wrap, and fastened to the body with adhesive but non-irritating tape.
- Time intervals for shavings or clipplings: 5 days a week for 4 weeks.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Application areas were cleaned with lukewarm water.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0, 10, 100 and 1000 mg/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not reported
- Amount(s) applied (volume or weight with unit): 4 ml/kg body weight
- Concentration (if solution): 0.5% (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80.

USE OF RESTRAINERS FOR PREVENTING INGESTION: No.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stock solutions of the test substance in acetonitrile with concentrations of 1 000 μg/ml, 205 μg/ml, and 200 μg/ml were prepared as follows: 100 mg, 20.5 mg, and 20 mg portions of test article were weighed into 100-ml volumetric flasks and dissolved in about 70 ml of acetonitrile by means of an ultrasonic bath. Afterwards, the volumetric flasks were filled to volume with HPLC-eluent. Next, various standard solutions were prepared by respective dilution of these stock solutions with HPLC-eluent to yield concentrations in the range from 5 μg/ml to 51.25 μg/ml. These standard solutions were used to calibrate the HPLC.

HPLC conditions:
- Apparatus: Merck L6200A (pump), Merck L4000 (UV-detector), Merck D2000 (integrator), Merck 655A 40 (sampling unit).
- Column: Lichrospher 100 RP-18; 5 μm; 125 x 4.6 mm (o.d.)
- Temperature: room temperature
- Eluent: Acetonitrile/0.02 M H3P04 (60+40/v/v)
- Flow: 1.0 ml/min
- Wavelength: 274 nm
- Injection volume: 10 μl

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days a week for 4 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 100 and 1000 mg/kg bodyweight
Basis:
nominal per unit body weight

No. of animals per sex per dose:

5 animals of each sex were used per dose.
- Group 1: 0 mg/kg
- Group 2: 10 mg/kg
- Group 3: 100 mg/kg
- Group 4: 1000 mg/kg

Control animals:

yes

Details on study design:

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): random

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily (including weekends)

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Following application 17 hours after removal of patches.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution, leukocyte count, neutrophils, eosinophils, basophils, lymphocytes, monocytes, large unstained cells, thrombocyte count, prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters checked: glucose, urea, creatinine, total bilirubin, total protein, albumin, globulin, A/G ratio, cholestrol, triglycerides, sodium, potassium, calcium, chloride, phosphorous inorganic, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At necropsy the following weights were recorded from all animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen.

The following organs and tissues were preserved in neutral buffered 4% formalin:
skin application site, skin remote site, mammary area, spleen mesenteric lymph node, axillary lymph node, sternum with bone marrow, femur with joint skeletal muscle, trachea, lung, heart, aorta, submandibular salivary gland, liver, pancreas, oesophagus, stomach, small intestine, large intestine, kidney (both), urinary bladder, prostate, seminal vesicle, testis (both), epididymis (both), uterus, vagina, ovary (both), pituitary gland, adrenal gland (both), thyroid with parathyroid gland, thymus, peripheral nerve, brain, spinal cord, eye with optic nerve (both), orbital gland (both), extraorbital lacrimal gland (both), Zymbal gland (both), muzzle, tongue, any tissue with gross lesions

HISTOPATHOLOGY: Yes
After the fixation, organ samples listed below were taken, embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin, and subjected to a microscopical examination: skin application site, skin remote site, liver, spleen, kidney (both), bone marrow, thymus, thyroid with parathyroid gland.

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Wilcoxon's two-sample test and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

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

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs or local irritation attributed to the treatment with test material. Two animals from the 1000 mg/kg dose group, one animal from the 100 mg/kg dose group, one animal from the 10 mg/kg dose group and two control animals developed blisters in the clipped areas of the back. As these findings were noted in treated and non treated animals, they were not attributed to treatment.

All animals survived the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Males treated at 100 and 1000 mg/kg body weight gained less body weight, compared to the control. However, since individual animals, which contributed mainly to this effect, had a lower body weight gain before treatment start (week -1), it was considered to reflect biological variability and was not attributed to the treatment. Throughout the study including the pretest week, the mean body weights of male group 2 were higher than that of the control group. This was also attributed to reflect biological variability.

FOOD CONSUMPTION
During week 2 of treatment increased mean food consumption was measured for females of group 4, which was, however, attributed to biological variability.

FOOD EFFICIENCY
Not examined

WATER CONSUMPTION
Not examined

OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
No significant findings.

Higher values were obtained for hemoglobin concentration distribution width (HDW) in male animals of group 4 (1000 mg/kg), however in the absence of other corroborative findings on red blood cell parameters no toxicological relevance was attributed. Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered andjor the values were within the reference range. Therefore, the following changes were considered not to be treatment-related: In group 2 (10 mg/kg), lower values of red cell volume distribution width (RDW) was noted in males, minimally higher levels of hemoglobin (Hb) and mean corpuscular hemoglobin concentration (MCHC) was observed in females. Females of group 3 (100 mg/kg) had higher white blood cell counts, particularly basophils, lymphocytes and large unstained cells. Males of group 4 (1000 mg/kg) showed minimally lower values of large unstained cells and females of this dose group had slightly lower platelet counts.

CLINICAL CHEMISTRY
In females of group 4 (1000 mg/kg), a minimal increase of plasma potassium concentrations was noted. Since no further alterations in electrolyte concentrations were observed in animals on this study, the relevance of this finding is equivocal. Lower plasma cholesterol concentrations as observed in females of group 4 are within the range of reference values and were therefore considered unrelated to the treatment.

Other minor deviations which attained a level of statistical significance occurred without any relation to the dose administered, and were therefore considered not to be treatment-related: In males of group 2 (10 mg/kg) changes of plasma creatinine concentrations and lower activities of alanine aminotransferase. In group 3 animals (100 mg/kg), decreased albumin to globulin ratios were noted in males, changes of plasma albumin concentrations, higher plasma triglyceride levels and higher activities of alanine aminotransferase and alkaline phosphatase were observed in females.

URINALYSIS
Not examined

NEUROBEHAVIOUR
Not examined

ORGAN WEIGHTS
No significant findings.

Minimal lower values of heart to body weight ratios were calculated for female groups 2, 3, and 4 (10, 100 and 1000 mg/kg body weight). In the absence of any corroborative findings and lack of dose dependency, these deviations were considered of incidental nature.

GROSS PATHOLOGY
One female (0 mg/kg) had a mottled thymus and one male (1000 mg/kg) presented a cyst in one kidney. These lesions were similar to those occurring spontaneously in our colony of rats. Thus no experimental relevance was attributed to these findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
No significant findings.

Changes found at the skin application site occurred in a similar incidence and morphological appearence in controls and treated animals. They were considered to be the result of the application procedure itself and not related to the treatment with the test article. Additionally, a variety of other changes was found in this study. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphologic appearance gave any indication of a treatment-related association.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No significant findings.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Analytical verification of doses:

The mean test article concentrations in the vehicle were 83.2%, 85.0% and 97.7% of the nominal concentrations in dose group 2, 3 and 4, respectively. The test material was found to be homogeneously distributed and to be stable in the vehicle under actual conditions of administration over the period of dosing.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the no-observable-effect level (NOEL) for rats of both sexes is 1000 mg/kg body weight.

Executive summary:

The study was performed according the requirements of OECD Guidelines for Testing of Chemicals No. 410 under GLP conditions. The study was performed to investigate the potential toxicity of the test material to male and female Tif: RAif (SPF), hybrids of RIII1 x RIII2 (Sprague-Dawley derived) rats when administered topically 5 days a week for 4 weeks. The dermal route was used as this represents a possible route of exposure in humans. Four groups of 5 male and 5 female rats were dosed with the test material in vehicle (0.5 % (w/v) carboxymethylcellulose in 0.1% (w/v) aqueous polysorbate 80) at 0, 10, 100 or 1000 mg/kg bw/day 5 times a week for 4 weeks. Test material was applied under occlusive dressing for a 6 hour period. Clinical observations, body weight and food consumption were recorded daily. Animals were humanely sacrificed at the end of the study. At termination, blood samples were taken for haematology and blood chemistry investigations. Selected organs were weighed and specified tissues/organs were processed for histopathological examination. Tissues/organs from control and high dose animals were examined by light microscopy, together with all macroscopic abnormalities in any group.

Analytical data confirmed the suitability of the dosing preparations used in the study. There were no adverse treatment-related effects on clinical observations, body weights, food consumption. There were no toxicologically significant effects on haematology, blood chemistry. There were no treatment-related macroscopic or microscopic findings in any organ at examination post mortem. The no effect level (NOEL) in this study was 1000 mg/kg bw/day.