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Toxicological information

Repeated dose toxicity: oral

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Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-15 until 2012-06-07
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non GLP 14 day dose range finding study for the subsequent Reproduction/Developmental Toxicity Screening Test

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
14 day dose range finder
GLP compliance:
not specified
, however the principles of GLP were followed and all data were recorded and retained
Limit test:

Test material

Test material form:
other: liquid

Test animals

other: Rat, RccHanTM: WIST(SPF)
Details on test animals and environmental conditions:
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Male animals: 369 - 397 g; Female animals: 172 - 227 g
- Fasting period before study: no
- Housing: before randomisation: 3 or 4 animals sex/cage in Makrolon Type IV cages; after randomisation individually in Makrolon Type III cages
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available
ad libitum in water bottles
- Acclimatisation period: Minimum of 5 days

- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
corn oil
Details on oral exposure:
The dose formulations were prepared daily using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The test item was formulated in the vehicle in concentrations of 0, 75 mg/mL, and 250 mg/mL. A constant treatment volume of 4 mL dose preparation/kg body weight was administered in all groups
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle to confirm the stability (8 days at room temperature (20 ± 5 °C)). The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to analysis laboratory.
Duration of treatment / exposure:
14 days
Frequency of treatment:
Once daily
Doses / concentrations
Doses / Concentrations:
0, 300 and 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
During the first 3 days of treatment, test item was administered by gavage. However, as a consequence of the unplanned deaths, microlabs were used from the fourth day of treatment onwards for application.
At the scheduled necropsy on day 15, animals were sacrificed by an injection of sodium pentobarbital.
Positive control:
Not applicable


Observations and examinations performed and frequency:
The following data were recorded on-line: clinical signs and observations, food consumption, body weights, necropsy findings and organ weights (TOX CONTROL).

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily during pre-treatment period, 3 times on first day of treatment, twice thereafter up to day of necropsy).
Food Consumption: Recorded for the following periods: during pretreatment period days 1 - 7; during the treatment period days 1 - 7 and 7 - 14.
Body Weights: Recorded for the following periods: once during pretreatment period; daily during treatment period.

Erythrocyte count
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Leukocyte count, total
Differential leukocyte count:
Platelet count

Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

Clinical Biochemistry
Bilirubin, total
Cholesterol, total
Aspartate aminotransferase
Alanine aminotransferase
Alkaline phosphatase
Bile acids
Protein, total
Albumin/Globulin ratio
Sacrifice and pathology:
At the scheduled necropsy on day 15, animals were sacrificed by an injection of sodium pentobarbital.

Any animal sacrificed or found dead during the study was weighed and subjected to macroscopic examination of all internal organs.
When considered appropriate, macroscopic changes in the animals were photographed and samples of tissue fixed in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.

Organ Weights
From all animals following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
Adrenal glands (weighed as pairs)
Kidneys (weighed as pairs)
Other examinations:
The following statistical methods were used to analyze food consumption, body weights, necropsy findings and organ weight:
• Means and standard deviations of various data were calculated.
• The Dunnett-test (many to one t-test) based on a pooled variance
estimate was applied if the variables could be assumed to follow a normal distribution
for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the
Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be
dichotomized without loss of information.

Results and discussion

Results of examinations

Details on results:
Analysis of Dose Formulations
Fatty acids, C18-unsatd. application formulations investigated during the study were found to comprise the test item in the range of 80.9% to 92.2%. All samples met the required content limit of ±20% with reference to the nominal concentration. The homogeneous distribution of Fatty acids, C18-unsatd. in the formulation was approved because single results did not deviate by more than 7.1% (<15%) from the corresponding mean. The application formulations were found to be not stable when stored for eight days at room temperature (20 ± 5 °C). Maximum variation from time zero (homogeneity) mean was 15.5% and therefore not within the required limit of 10%.

Viability / Mortality
There were no test-item related deaths.
Two males in the control group and at 300 mg/kg bw/day, respectively, two females at 100 mg/kg bw/day and one female at 300 mg/kg bw/day died due to an intubation error. One male at 300 mg/kg bw/day and one female at 100 and 300 mg/kg bw/day, respectively, were added to the study as a consequence of those deaths to add to the number of animals per group that achieve a 14-day treatment.

Clinical Signs or Observations
No test item-related clinical signs were noted during the whole course of the study. Clinical signs in the surviving animals were limited to slightly decreased spontaneous activity in one male of group 3 (1000 mg/kg) on the first day of treatment.

Food Consumption
There were no differences in mean food consumption between control animals and animals treated with Fatty acids, C18-unsatd.

Body Weights
There were no differences in mean body weights and mean body weight gain between control animals and animals treated with Fatty acids, C18-unsatd.

Clinical Laboratory Investigations

At 1000 mg/kg bw/day in males, the level of platelets was statistically significantly higher and in females statistically significantly lower than in the control group. The value for males was in the range of the historical control data (802 - 1260 G/L) but not for females (924 - 1695 G/L).
Nevertheless, these opposite results were considered to be incidental. No other findings were noted during assessment of the hematology data.

Clinical Biochemistry
No test-item related changes were noted in clinical biochemistry parameters. At 1000 mg/kg bw/day in males, the level of alanine aminotransferase was statistically significantly higher than in the control group. This value was in the range of the historical control data (33.3 - 77.3 U/L) and therefore not test item-related.

Organ Weights

No effects on organ weights were noted for males.
For males at 1000 mg/kg bw/day, absolute and relative (to body weight) liver weights were statistically significantly increased.
For females, organ weights were similar between dose groups and controls.

Macroscopical Findings
No test item-related macroscopical findings were noted at any dose level.
One male at 1000 mg/kg bw/day showed a pelvic dilation of the kidneys and one female at 300mg/kg bw/day dilation with watery fluid of both uterus horns and a nodule in the cervix.
Furthermore, the same animal had a firm dark red and yellowish nodule of the adipose tissue in the body cavity. Of the five animals that died as a consequence of intubation error during the first 3 days of treatment, two showed incompletely collapsed lungs with a dark red discoloration.

Effect levels

Dose descriptor:
Based on:
test mat.
Basis for effect level:
other: 14 day dose range finding study oral
Remarks on result:
not determinable
no NOAEL identified

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion