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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
2,5,7,10-tetraoxaudecane
IUPAC Name:
2,5,7,10-tetraoxaudecane
Details on test material:
- Name of test material (as cited in study report): 2,5,7,10-tetraoxaudecane (TOU)
- Physical state: liquid
- Analytical purity: 99.632%
- Impurities (identity and concentrations): water (0.142%); aldehydes (not found); alcohols (0.064%)
- Lot/batch No.: 0804061200
- Expiration date of the lot/batch: 06/04/2011
- Storage condition of test material: room temperature

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CER-Group Dépt. Santé (Marloie, Belgium)
- Age at study initiation: 12 weeks
- Weight at study initiation: 2-3 kg
- Fasting period before study: no
- Housing: During on-study periods, the rabitts were housed individually in cages in the animal facilities of CER-Dept. Santé at Marloie (B6900), Belgium.

- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
The water quality was in compliance with the Belgium Water Supply (Water quality) Regulations. Routine chemical and bacterial analyses are conducted periodically by the local water authority.
- Acclimation period: The animals were housed 5 days in the experimental unit before use on the study. During acclimatisation, animals were daily fed with maintenance diet for rodent and had a free access to tap water ad libitum in bottles.

ENVIRONMENTAL CONDITIONS
Temperature and relative humidity were recorded daily during the administration and study periods.
- Temperature (°C): 18 ± 5
- Humidity (%): 28-60
- Air changes (per hr): 10-30
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: dorsal area of the trunk
- % coverage: approximately 10% of the total body surface
- Type of wrap if used: non-irritant tape
- Time intervals for shavings or clipplings: The clipping was repeated at different interval (weekly) in order to ensure optimal contact between the test item and the animal skin. The clipped fur area for test item application corresponded of about 10 percent of the total body surface area of animals.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): not specified
- Time after start of exposure: not specified

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
For each animal a syringe was filled with an appropriate volume of dosing solution. The syringe with its canula was weighed and the weight noted on the prepared table. After application of the dosing solution on the dorsal area of the trunk, the empty syringe with its canula was weighed and the weight noted. The exact injection time was also recorded. A range of 15% in regard with the theoretical dose (100, 400 or 1000 mg.kg-1) was judged as acceptable for the administered test item dose.
- Concentration (if solution): 100, 400, 1000 mg/kg
- Constant volume or concentration used: yes (A range of 15% in regard with the theoretical dose (100, 400 or 1000 mg.kg-1) was judged as acceptable for the administered test item dose)


VEHICLE: none


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Weight of full and empty syringe were measured to determine weight of applied test item and thus dose of applied test item each day of administration for each animal, for each dose.
Duration of treatment / exposure:
28 days
Frequency of treatment:
5-day per week basis, 6h per day
(Days 1, 2, 3, 6, 7, 8, 9, 10, 13, 14, 15, 16, 17, 20, 21, 22, 23, 24, 27, 28)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
400 mg/kg bw
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal per unit body weight
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The randomization procedure of animals was performed according to the SOP QA/GEN18-31. Randomization was performed at the beginning of the acclimatization period under supervision of the QA manager of the Test Site for the animal experiments. The computer randomization was performed according to the following procedure: 20 rabbits were divided in four groups consisting of 2 males and 2 females, and with an average weight equivalent between groups.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were clinically examined by a veterinarian at randomisation, one day before test item application and weekly during toxicity trial period. Clinical

BODY WEIGHT: Yes
- Time schedule for examinations: Individual weights of animals were determined at the day of test item application and weekly thereafter

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all animals
- Parameters checked in tables 6, 33 to 45 in the attached report were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No data
- How many animals: all animals
- Parameters checked in tables 6, 33 to 45 in the attached report were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
All animals included in the toxicity study (control and treatment groups) were subjected to gross necropsy which included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. For possible future histopathological examination, different organs and tissues were collected from each rabbit by the staff of the Test Site and preserved in a suitable medium (4 percent buffered formalin). After euthanasia by CO2 narcosis, the following organs were sampled from each rabbit: skin (both treated and adjacent untreated areas),
liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, heart, encephalon, spinal cord, stomach, small intestine, large intestine, trachea, lung, gonads, sexual organs, bladder, lymph node, peripheral nerve (sciatic), bone marrow.
Statistics:
At the end of this period, a clinical examination was realized in all animals. The results of different blood analysis parameters were analyzed from a statistical point of view by means of the software Statistica version 7.1 Fr.
In order to compare the means of two groups, the t-test (parametric test) was used because it is employed even on samples of small size (n=10). The t-test assesses whether the means of two groups are statistically different from each other.
The parametric tests are performed when the variables investigated are normally distributed and when there is homogeneity of variances. A preliminary test is used to check this last condition (test of Fisher). If the P variances obtained with this test is inferior to 0.05, the homogeneity of variances is not respected, nonparametric tests are then carried out.
The significance criterion is fixed at 0.05 in this study. If the P value is lower than 0.05, the difference in mean for the control group and the treated group is statistically significant.

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:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
Animals have shown no response to the administration of the test item at the post-injection observation.
Individual weights of animals were determined at the day of test item application and weekly thereafter. Mean animal body weights per treatment group are listed in Table 4. No significant weight changes were highlighted in the time-course of study.
All animals were clinically examined by a veterinarian at randomisation, one day before test item application and weekly during toxicity trial period. Clinical examinations included a general examination and following parameters were checked: central nervous system (CNS), respiratory, gastrointestinal (GI), skin, circulatory, eye, skeletomuscular and urogenital systems, temperature and body weight. The results of the clinical examination were reported on the corresponding forms.

Controls and treated animals appeared in good health during the treatment period trial and no injuries or severe signs of toxicity were observed during the daily application of the test item at a final dose of 100, 400 or 1000 mg/kg.
Only a lack of skin elasticity resulting from a repeated application of the test item has been demonstrated in the treated animals. This observation has been highlighted on the seventh day of application in the 400 and 1000 mg/kg treatment groups. On the eighth day of treatment, this lack of
skin elasticity was observed in all treatment groups (100, 400 or 1000 mg/kg).
The lack of skin elasticity was not accompanied by redness or other skin problems and was restricted only to the area of test item application. The animals did not suffer of this injury.
Daily application of test item (100, 400 or 1000 mg/kg) over a 28-days period has no induced morbidity or mortality in animals even in the group treated with the highest dose which correspond to the dose level of the limit-test like recommended by the OECD guideline 410.
From these results and observations, it appears that the maximum dose of 2,5,7,10-tetraoxaundecane that causes no detectable adverse effects (alteration of morphology, functional capacity, growth, development or life span) in rabbit and under the defined conditions of this experiment is 1000 mg per kg per day. This dose corresponds to the NOAEL (No Observed Apparent Effect Level) of this experimentation.
At the end of the study period, clinical and gross necroscopy examinations were performed.

CLINICAL EXAMINATIONS
Clinical examinations included haematology (red blood cell count, haemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count and platelet count) and clinical blood chemistry
(calcium, phosphorus, chloride, sodium, potassium, fasting triglycerides, acid/base balance, methaemoglobin and cholinesterase activity. Parameters of liver and kidney function: serum aspartate aminotransferase, serum alanine aminotransferase, albumin, blood creatinine, total bilirubin, uremia
and total serum protein).
All animals included in the toxicity study (control and treatment groups) were subjected to gross necropsy which included examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents. For possible future histopathological examination, different organs and tissues were collected from each rabbit by the staff of the Test Site and preserved in a suitable medium (4 percent buffered formalin). After euthanasia by CO2 narcosis, the following organs were sampled from each rabbit: skin (both treated and adjacent untreated areas),
liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, heart, encephalon, spinal cord, stomach, small intestine, large intestine, trachea, lung, gonads, sexual organs, bladder, lymph node, peripheral nerve (sciatic), bone marrow.
The liver, kidneys, brain, spleen, epididymides, ovaries, thymus, heart, adrenals and testes were weighed wet immediately following dissection to avoid drying. These data were recorded on a special sample collection form and signed by the executing technician.
Each tissue sampled was labelled with the study number, animal number, name of the group and the date of sampling.

ANATOMOPATHOLOGICAL EVALUATION
The following organs were processed for histology and examined microscopically:
• control and 1000 mg/kg groups: brain, epididymides, kidneys, heart, liver, lungs, ovaries, skin (untreated area), testes, urinary bladder and uterus,
• 1000 mg/kg group only: skin (application site).

Treatment-related effects were observed at the application site in all animals treated at 1000 mg/kg. There were characterized by slight epidermal hyperplasia, minimal to slight orthokeratotic hyperkeratotis and slight superficial perivascular inflammation. In addition, slight epidermal erosion/ulcer was present in 2/5 treated males at 1000 mg/kg. Minimal epidermal hyperplasia and/or minimal orthokeratotic hyperkeratotis were also recorded in the untreated skin area of 1/5 males and 2/5 females treated males at 1000 mg/kg.
Evidence of urolithiasis were observed in the urinary bladder of 3/5 treated males at 1000 mg/kg. Urolithiasis is known to occur commonly in rabbits (Hoefer, 2006) and in the absence of any treatment related effects on mineral deposition in other organs, this occurrence was considered to be fortuitous.
All other microscopic findings were those commonly seen in rabbits and bore no relationship to treatment.

The microscopic findings at the application site were indicative of slight local dermal irritation of 2,5,7,10-tetraoxaundecane. The epidermal hyperplasia and orthokeratotic hyperkeratotis recorded in the untreated skin area of a few treated animals were probably due to application procedure and/or local diffusion of the test article.

The dermal application of 2,5,7,10-tetraoxaundecane (at least 6 hours per day on 5-day per week basis) for 28 days in rabbits at the concentration of 1000 mg/kg induced histological evidences of slight irritation at the application site.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
dermal irritation
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Daily application of test item (100, 400 or 1000 mg/kg) over a 28-days period has no induced morbidity or mortality in animals even in the group treated with the highest dose which correspond to the dose level of the limit-test like recommended by the OECD guideline 410.
From these results and observations, it appears that the maximum dose of 2,5,7,10-tetraoxaundecane that causes no detectable adverse effects (alteration of morphology, functional capacity, growth, development or life span) in rabbit and under the defined conditions of this experiment is 1000 mg per kg per day. This dose corresponds to the NOAEL (No Observed Apparent Effect Level) of this experimentation.
Executive summary:

Daily application of test item (100, 400 or 1000 mg/kg) over a 28-days period has no induced morbidity or mortality in animals even in the group treated with the highest dose which correspond to the dose level of the limit-test like recommended by the OECD guideline 410.

From these results and observations, it appears that the maximum dose of 2,5,7,10-tetraoxaundecane that causes no detectable adverse effects (alteration of morphology, functional capacity, growth, development or life span) in rabbit and under the defined conditions of this experiment is 1000 mg per kg per day. This dose corresponds to the NOAEL (No Observed Apparent Effect Level) of this experimentation.

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