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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
EC Number:
601-478-9
Cas Number:
117428-22-5
Molecular formula:
C18H16F3NO4
IUPAC Name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
Test material form:
solid
Details on test material:
- Purity: 93.3-99.8% (see individual test record for specific details)
Specific details on test material used for the study:
99.3% purity

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the test facility.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 58 days (males); approximately 59 days (females)
- Weight at study initiation: 255.1-294.7 g (males); 174.9-221.1 g (females)
- Housing: Animals were housed singly in stainless steel, wire-mesh cages suspended above cage boards. Enrichment (nylabone toy) was placed in each cage. Each cage rack contained only animals of one sex.
.- Diet (e.g. ad libitum): ad libitum, except when rats were fasted
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C (64-79°F)
- Humidity (%): 30-70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
The test material was moistened with water to form a thick paste
Details on exposure:
Approximately 24 hours prior to the first treatment, the fur of each rat was closely shaved to expose the skin from the back and trunk. The target area to be treated (5 cm x 7.4 cm = 37 cm2) was marked on the back of each rat with a water-insoluble marker. The 37 cm2 treatment area is approximately equal to 10% of the total body surface area for rats in the 200 to 300 g range of body weights.

The test substance was moistened with deionized water to form a thick paste and applied in a thin and uniform layer to cover as much of surface of the target area as possible. The test site was covered with a 2-ply porous gauze pad followed by successive layers of stretch gauze (no more than 8 layers) and self-adhesive bandage. The control rats were treated with deionized water at the same volume as the high-dose rats. Some rats were fitted with plastic collars during the exposure period to prevent disruption of the wrappings. The rats were checked for dislodged wrappings several times daily during the exposure period.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
29 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
300 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 0, 100, 300, and 1000 mg/kg/day were selected for this study. The 1000 mg/kg/day dosage is a limit dose level. The other dosages were selected to establish a no-observed-adverse-effect level (NOAEL) and to assess a dose response for any observed effects.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cage-site examinations to detect moribund or dead rats and abnormal behavior and/or appearance among rats were conducted at least twice daily throughout the study. Abnormal behavior/appearance was recorded by exception.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before dosing on test day 0 and then weekly thereafter soon after test substance removal, each rat was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all rats. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior. Any abnormal clinical signs noted were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The rats were weighed on test day 0 and at weekly intervals during the study. All rats were weighed on the day of sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; The amount of food consumed by each rat was determined weekly by weighing each feeder at the beginning and end of the interval and subtracting the final weight and the amount of spillage from the feeder during the interval from the initial weight. Food spillage < 5 g was not included in the calculation. From these measurements, mean daily food consumption over the interval was determined.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: Yes; calculated from food consumption and body weight data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes; Two ophthalmology examinations were conducted by a veterinary ophthalmologist. The pretest examination was performed on all rats received for the study, prior to assignment to groups. Two rats with preexisting ophthalmology abnormalities were eliminated from consideration for use in the study. Surviving rats were examined prior to the final sacrifice. Both eyes of each rat were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Yes; carbon dioxide
- Animals fasted: Yes
- How many animals: All
- Parameters examined: red blood cell count, red cell distribution width, hemoglobin, absolute reticulocyte count, hematocrit, platelet count, mean corpuscular (cell) volume, white blood cell count, mean corpuscular (cell) hemoglobin, differential white blood cell count, mean corpuscular (cell) hemoglobin concentration, microscopic blood smear examination, prothrombin time, and activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Animals fasted: Yes
- How many animals: All
- Parameters examined: aspartate aminotransferase, glucose, alanine aminotransferase, total protein, sorbitol dehydrogenase albumin, alkaline phosphatase, globulin, total bilirubin, calcium, urea nitrogen, inorganic phosphorus, creatinine, sodium, cholesterol, potassium, triglycerides, and chloride.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters examined: quality, ketone, color, bilirubin, clarity, blood, volume, urobilinogen, specific gravity (SG), protein, pH, microscopic urine sediment examination, and glucose.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following tissues were collected from all of the rats:
Cardiovascular System: Heart and aorta
Digestive System: Liver, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum, salivary glands, and pancreas
Endocrine System: Pituitary gland, thyroid gland, parathyroid glands, and adrenal glands
Female Reproductive System: Ovaries (with oviducts), uterus (including cervix), mammary glands, and vagina
Hematopoietic System; Spleen, thymus, mandibular lymph node, mesenteric lymph node, bone marrow, and Peyer’s patches
Male Reproductive System: Testes, epididymides, prostate, and seminal vesicles with coagulating glands
Miscellaneous: Skin (treated and adjacent material), skin adjacent to the mammary gland, eyes (including retina and optic nerves), and gross observations
Musculoskeletal System: Skeletal muscle, femur/knee joint, and sternum
Nervous System: Brain (including cerebrum, midbrain, cerebellum, and medulla/pons), spinal cord (cervical, mid-thoracic, and lumbar sections), and sciatic nerve
Respiratory System: Lungs, trachea, nose (4 sections), larynx, and pharynx
Urinary System: Kidneys and urinary bladder

The following tissues were weighed from rats sacrificed by design at the end of the study: liver, kidneys, heart, spleen, thymus, adrenal glands, brain, testes, epididymides, accessory sex organs (prostate + seminal vesicles with coagulating glands), ovaries (with oviducts) and uterus (including cervix). Paired organs were weighed together. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated. Testes, epididymides, and eyes were fixed in modified Davidson’s solution. All other tissues were fixed in 10% neutral buffered formalin. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist. All collected tissues from rats in the control and 1000 mg/kg/day groups, and tissues collected from early decedents were processed to slides and evaluated microscopically.
Statistics:
For Body Weight, Body Weight Gain, Food Consumption, Food Efficiency, Clinical pathology,and Organ weights, use Levene’s test for homogeneity and the Shapiro-Wilk test for normality; If the preliminary test is not significant, use the One-way analysis of variance test followed by Dunnett’s test; If the preliminary test is significant, use the Kruskal-Wallis test followed by Dunn’s test. Significance was judged at p < 0.05. Separate analyses were performed on the data collected for each sex.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of systemic toxicity were observed. Incidental incidences of ocular or nasal discharge, scabs, wounds, wet fur, paleness, and stained skin/fur were not considered to be test substance related as the clinical signs were observed in both treated and control animals and/or the incidence did not occur in a dose-related pattern. Swollen head or nose observed in several animals was considered to be due to the wrapping procedure.
Dermal irritation:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the course of the study, one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group died on test days 14, 9, 9, and 13, respectively. The deaths of these rats were considered to be a result of the wrappings being too tight.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related effect was observed on mean body weight in any male or female group. Mean body weights on test day 28 in the 1000 mg/kg/day groups were 101% and 98% of control (neither statistically significant) for males and females, respectively.

No adverse or test substance-related effect was observed on mean body weight gain in any male or female group. Mean overall (test days 0-28) body weight gain in the 1000 mg/kg/day male group was 101% of control (not statistically significant). Mean overall body weight gain in the 1000 mg/kg/day female group was 80% of control. This decrease in body weight gain was considered to be non-adverse because the decrease was not statistically significant compared to control and was driven by the low body weight gain of one animal.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test substance-related effect was observed on mean overall food consumption (test days 0-28) in any male or female group. The mean overall food consumption in the 1000 mg/kg/day groups was 102% and 99% of control (neither statistically significant) for males and females, respectively.
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
No adverse or test substance-related effect was observed on mean overall food efficiency (test days 0-28) in any male or female group. The mean overall food efficiency in the 1000 mg/kg/day male group was 99% of control (not statistically significant). The mean overall food efficiency in the 1000 mg/kg/day female group was 83% of control (not statistically significant). This decrease correlated with the decreased body weight gain in that group and was considered to be non-adverse.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmological changes were noted in male or female rats at any dose level.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or adverse changes in group mean haematology parameters at test day 29 in male or female rats. The following statistically significant changes in mean haematology parameters were not adverse or not related to exposure to the test substance as the change did not occur in a dose-related pattern:
• Platelet count (PLT) was minimally increased in male rats dosed with 300 mg/kg/day (153% of control).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean clinical chemistry parameters at test day 29 in male or female rats. The following statistically significant changes in mean clinical chemistry parameters were considered to be potentially treatment related but non-adverse:

• Alanine aminotransferase (ALT) was minimally decreased in male and female rats at 100, 300, or 1000 mg/kg/day (variable statistical significance). These changes were minimal, were not clearly dose related, and there were no correlative changes in other associated liver enzyme parameters (AST, SDH, BILI) or liver histology. Therefore, the decreases in ALT in both male and female rats were considered to be potentially treatment related but non-adverse.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There were no adverse changes in group mean urinalyses parameters at test day 29 in male or female rats. The following statistically significant change in a mean urinalyses parameter was considered treatment related but non-adverse:

• pH was minimally decreased in female rats dosed with 1000 mg/kg/day (92% of control). Although individual urine pH values of only three female rats in the 1000 mg/kg/day group were lower than the lowest value observed in control rats (pH 6.0), they were not considered to be outside the range that is normally observed in female rats of this age and strain (95% CI for female rat urine pH 6.0 - 7.5). Therefore, this minimal change was considered to be treatment related but non-adverse.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on organ weights. The following statistically significant changes in the organ weight were considered non-adverse or unrelated to exposure to compound.

In males, mean absolute and mean relative (% brain weight) adrenal weights were increased 122% and 121% in the 1000 mg/kg/day exposure group, respectively, as compared to control values. Because these adrenal weight changes were not associated with changes in adrenal weight relative to body weight or with correlative microscopic findings, and because no adrenal weight changes occurred in female rats at any dose, they were considered spurious and unrelated to test substance administration.

In females, mean relative (% body weight) liver weight was increased 111% in the 1000 mg/kg/day exposure group as compared to the control value. Because the liver weight change was not associated with changes in absolute liver weight or relative to brain weight or with correlative microscopic findings, and because no liver weight changes occurred in male rats at any dose, this minor change in mean relative liver weight was considered spurious and unrelated to test substance administration.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All gross observations at the terminal necropsy were consistent with normal background lesions in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test substance-related microscopic findings. Microscopic changes likely occurring secondary to the wrapping procedure were present in the liver of a few rats across all the groups.

In a few animals across all the groups, including controls, there were microscopic observations in the liver, which were diagnosed as mild to moderate focally extensive necrosis. The lesion was characterized by focally extensive to multifocal areas of coagulative necrosis variably admixed with chronic inflammation. The lesion was primarily observed beneath the capsular surface but was also noted to extend into the hepatic parenchyma. The lesion was usually limited to one lobe only. In one animal, which died on test day 9, bridging necrosis accompanied with mixed inflammatory cell infiltration was noted.

These liver changes are similar to those previously reported to occur in rats on dermal studies. In those studies, the subcapsular liver changes were attributed to pressure on the liver by the abdominal wrap. Therefore, based on the morphology of the liver lesions observed in the current study, and their occurrence in both treated and control animals, these changes were considered to be secondary to the wrapping procedure and not primary test substance-related effects.

All other microscopic findings were consistent with normal background lesions in rats of this age and strain.
Other effects:
no effects observed
Description (incidence and severity):
There were no statistically significant or treatment-related changes in coagulation parameters at test day 29 in male or female rats.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.
Executive summary:

The objective of this study was to determine the potential toxicity of the test substance following repeated dermal exposure. The test substance was applied to the shaved, intact skin of male and female Crl:CD(SD) rats for 6 hours/day for 29 consecutive days. Three groups of 10 male and 10 female rats were treated dermally with 100, 300, or 1000 mg/kg/day of the test substance. A control group of 10 male and 10 female rats was similarly treated with deionized water. Body weights, food consumption, and detailed clinical observations were evaluated weekly. The animals were examined for acute clinical signs of systemic toxicity on days detailed clinical observations were not conducted. Ophthalmological assessments were performed prior to the start of exposure and near the end of the exposure period. Clinical and anatomic pathology endpoints were evaluated at the end of the exposure period.

 

No test substance-related deaths occurred. The deaths of one male rat each in the 100, 300, and 1000 mg/kg/day groups and one female rat in the 300 mg/kg/day group were attributed to their wrappings being too tight. No clinical signs of systemic toxicity were observed. No adverse or test substance-related effects occurred on body weights, body weight gains, food consumption, or food efficiency for any male or female dose group. No ophthalmological lesions were observed.

 

There were no test substance-related adverse effects on clinical pathology parameters, organ weights, gross pathology, or microscopic pathology.

 

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for male and female rats was 1000 mg/kg/day, the highest dosage tested. This NOAEL is based on the absence of adverse effects on in-life, clinical pathology, and anatomic pathology parameters at any dosage tested.