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Repeated dose toxicity: dermal

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Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
other: dose range-finding study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: non-GLP dose range finding study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
The study is a dose range-finding study for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats.
GLP compliance:
no
Remarks:
Study is a Dose Range Finding Test (DRFT)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear liquid
Details on test material:
- Name of test material (as cited in study report): Isononyl Isononanoate
- Physical state: liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: males 308 t0 340 g, females 211 to 249 g
- Fasting period before study: no
- Housing: all animals were housed individually in clean, stainless steel wire mesh cages
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum municipal water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2 to 22.4
- Humidity (%): 35.8 to 42.2
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 11 December 2012 To: 21 December 2012

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 10% of body surface
- Type of wrap if used: gauze and flexible bandages (Co-Flex® or PetFlex®) (first 7 dermal applications only)
- Time intervals for shavings or clipplings: prior to initiation of dose administration and as often as needed thereafter

REMOVAL OF TEST SUBSTANCE
- Washing (if done): If needed, the test site was gently patted with gauze moistened with sterile water and then patted again with dry gauze or a dry disposable paper towel.
- Time after start of exposure: 6 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.59, 1.18 and 2.35 mL/kg

VEHICLE : no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, Elizabethan style collars during the 6-hour exposure
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Homogeneity and concentration assessments were not conducted on this non-GLP range finding study because the test item was applied pure with no dilution.
Duration of treatment / exposure:
10 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw/day
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
other: yes, application of sterile water
Details on study design:
- Dose selection rationale: Exposure levels were provided by the Sponsor Representative after consultation with the Study Director. A repeat-dose dermal study using this test item has not previously been conducted in rats. It was not expected that the test item would be absorbed at levels necessary to result in excessive systemic toxicity. Because this is a dose range-finding study for a test item not previously tested by repeated dermal application, there was the inherent possibility that systemic toxicity or local skin irritation would occur.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily; each male and female was also observed for signs of toxicity approximately 1 hour following each dose administration.

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: The application site was scored daily (prior to dose administration during the treatment period).

BODY WEIGHT: Yes
- Time schedule for examinations: on study days 0, 4, 7, and 10

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: No
Other examinations:
organ weights
adrenal glands, ovaries with oviducts, brain, spleen, epididymides,testes, heart, thymus gland, liver, kidneys, thyroids with parathyroids
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-exposed group to the control group by sex.
Mean body weights, body weight changes, food consumption, and absolute and relative organ weights were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item exposed groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
vocalization 1 high dose female
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
erythema, scabbing, desquamation and fissuring; exfoliation in females only
Mortality:
mortality observed, treatment-related
Description (incidence):
vocalization 1 high dose female
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lower in high dose animals
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
lower in high dose males
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):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
thymus, spleen and adrenal gland
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: vocalization upon handling was noted for 1 high dose female during study days 5 to 10, which was attributed to very slight to moderate erythema, very slight to slight edema, fissuring and desquamation at the dermal application site.
One mid dose female was found dead approximately 45 minutes following administarion of the first dose. The cause of death was not test substance related.

DERMAL OBSERVATIONS: at the dermal application site very slight to moderate erythema, very slight to slight edema, scabbing and desquamation was noted in animals of all dose groups as early as study day 3 and generally continued till the end of the study. The incidence and severity of the findings were similar across groups. No change in incidence and severity of edema, erythema or desquamation was observed after occlusion was discontinued on study days 7 to 10. Fissuring was observed in females in de low dose group (2f) and in 2-4 males and females in the mid and high dose groups during study days 4 to 10. After occlusion of the dermal site was discontinued on days 7 to 10, the number of animals with fissuring at the dermal application site was slightly higher (8 out of 13 animals). Exfoliation at the dermal application site was observed during study days 9 and 10 for 1 and 3 females in the low and high dose), respectively.

BODY WEIGHT AND WEIGHT GAIN: During study days 0 to 4 high dose males showed a statistically significant mean body weight loss whereas high dose females showed absence of mean body weight gain. During study days 4 to 7 mean body weight gain was similar to the control group for males but slightly lower for the females. During study days 7 to 10 high dose males and females showed statistically significant mean body weight losses. Overall high dose mean body weight were statistically significant lower compared to the control group at the end of the study: 13.5% (males) and 13.1% (females). Animals in the low and mid dose groups showed mean body weight losses or no mean body weight gain only during study days 7 to 10. Overall, mean body weights were not statistically significant lower compared to controls on study day 10.

FOOD CONSUMPTION: mean food consumption (g/animal/day and g/kg/day) was statistically significantly lower in high dose males compared to control males during study days 0 to 4. Thereafter mean food consumption was similar to the control group.

ORGAN WEIGHTS: mean absolute and relative thymus weights were (not statistically significant) lower in all dose groups compared to the control group. In addition, mean absolute and relative spleen weights for low dose males and mid and high dose males and females were lower compared to the control group. High dose females showed also statistically significant higher mean absolute and relative adrenal gland weights.

Effect levels

Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Erythema and edema at the dermal application site in all test groups; changes in selected organ weights observed in all dose groups.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Dermal application to rats of isononyl isononanoate at doses of 0, 500, 1000 and 2000 mg/kg bw/day (no vehicle) resulted in fissuring, scabbing and desquamation in all dose groups, with vocalization of 1 high dose female. In all dose groups erythema and edema were noted at the dermal application site, with low severity. High dose animals showed statistically significantly lower mean body weights. In addition, changes in selected organ weights (spleen, thymus, adrenal gland) were observed in all dose groups. During days 7 to 10 when occlusion of the dermal application site was discontinued, the severity of the effects was more marked.
Based on the effects observed at all dose levels, a NOAEL could not be derived, and the LOAEL in this range-finding study is 500 mg/kg bw/day.