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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March - 23 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to GLP and OECD guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Rape oil, oxidized
EC Number:
305-871-3
EC Name:
Rape oil, oxidized
Cas Number:
95193-59-2
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Rape oil, oxidized
Details on test material:
- Name of test material (as cited in study report): Blown rapeseed oil
- Substance type: Yellow to brown viscous liquid
- Physical state: liquid
- Analytical purity: treated as 100% pure
- Expiration date of the lot/batch: 16 December 2010
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark
-Other: Specific Gravity 0.9660 g/cm3 (20°C)
-Other: pH (1% in water, indicative range) 4.9-4.7 (determined at NOTOX)

Test animals

Species:
rat
Strain:
other: Wistar-Han
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F0 Charles River Laboratories France, L'Arbresle Cedex. France.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: no data
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. Certificates of analysis were examined and then retained in the NOTOX archives. During activity monitoring, animals were housed individually in Macrolon plastic cages (MIII type; height 15 cm) with sterilized sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.2 – 21.4°C
- Humidity (%): 23 - 89%
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light and 12 hours darkness per day.
Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity (with a maximum of 4 hours). Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

IN-LIFE DATES: From: 09 March 2010 To: 23 April 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the test substance and vehicle.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Concentration in vehicle: 75, 225 and 500 mg/ml
- Amount of vehicle (if gavage): 2 mL/kg body weight
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No analyses were conducted since no analytical method could be developed for formulations of the test substance in corn oil.
Duration of treatment / exposure:
Exposure period: Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 69 (Group 3) and 73 (Group 4) were not dosed during littering.
Frequency of treatment:
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study (NOTOX Project 493033)
-Results of the dose range finding study are described in End point study record Repeated dose toxicity: oral.Notox Project 493025.
-5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology:
Males: the first 5 males per group
Females: with live offspring
- Post-exposure recovery period in satellite groups: none
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early morning/late afternoon)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made in all animals, immediately after dosing. Once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually. On March 30th 2010, no arena observations were performed for the males, but sufficient data was available from other observations.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4. No body weight was determined during the post-coitum period for female no. 46 (Group 1) because mating of this female was overlooked. Sufficient data was available for an accurate evaluation.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation. No food consumption was determined during the post-coitum period for female no. 46 (Group 1) because mating of this female was overlooked. Sufficient data was available for an accurate evaluation.

FOOD EFFICIENCY: Yes
- (food comsumption per animal per day/ average body weight per cage) * 1000

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group (females used had live offspring)
- Parameters checked were: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes,
eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Clotting Potential, Prothrombin time, Activated Partial thromboplastin time
Because of a possible switch of blood samples from female no. 45 (Group 1) and female no. 54 (Group 2) the rults for haematology and clinical biochemistry were excluded. These data were excluded and sufficient data from other animals is available to make an accurate evaluation.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Animals fasted: Yes, but water was provided
- How many animals: 5 animals/sex/group
- Parameters checked were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids
Because of a possible switch of blood samples from female no. 45 (Group 1) and female no. 54 (Group 2) the rults for haematology and clinical biochemistry were excluded. These data were excluded and sufficient data from other animals is available to make an accurate evaluation.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, motor activity:

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):

From the selected 5 animals/sex/group:
Identification marks (not processed), Adrenal glands, All gross lesions, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides (2), (Esophagus), Eyes (with optic nerve (if detectable) and Harderian gland; 2), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung, infused with formalin, Lymph nodes (mandibular, mesenteric), (Nasopharynx), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, (Skin), Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes (2), Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina

All remaining animals and females which failed to deliver**:
All gross lesions, Cervix, Clitoral gland, Coagulation gland, Epididymides*, Identification marks (not processed), Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes*, Uterus, Vagina

* Fixed in modified Davidson's solution (prepared at NOTOX using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.
** In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique (Ref. 1) in order to detect any former implantation sites (Salewski staining prepared at NOTOX using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

Four females (nos. 59, 60, 68, 78) were fasted approximately 3 hours longer than the maximum of 20 hours allotted before necropsy. The fasting period was only slightly longer and was considered not to have adversely affected the study's integrity.

HISTOPATHOLOGY: Yes
The delegated phase was performed by the Principal Investigator for histopathology.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
- The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all animals that failed to mate, conceive, sire or deliver healthy pups (female no. 57 and male no. 17, Group 2).
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test, Dunnett 1955) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test; Miller 1981) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

The number of implantation sites was subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences followed by the Wilcoxon test (Wilcoxon, 1945) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.

Incidental findings that were noted included scabbing of the left cheek and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.

The statistically significant increase in relative food consumption seen for males of at 450 mg/kg (Group 3) was not considered to be toxicologically relevant. The higher relative food consumption occurred in the absence of a dose-related distribution, and if treatment related toxicity were evident, a reduction in food consumption would be expected to be seen.

Very low food consumption was seen for female nos. 45 (Group 1) and 61 (Group 3) over Days 0-4 of the post coitum period, however, the low weights were transient and body weight gain for both of these animals was normal over the same period, thus the low values were not considered to be adverse.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Description (incidence and severity):
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with Blown rapeseed oil.

The statistically significant decrease in the activated partial thromboplastin time (APTT; males) at 1000 mg/kg compared to controls was not considered to be toxicologically relevant as the values for concurrent controls were slightly high and values at 1000 mg/kg remained within the range considered normal for rats of this age and strain.

The increase in neutrophil counts with concurrently reduced lymphocyte counts seen for animal no. 32 (Group 4, 1000 mg/kg) were not considered to be toxicologically relevant as it occurred only for an individual animal. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and not to be treatment related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

The significantly higher inorganic phosphate level seen for females at 150 mg/kg was considered to be of no toxicological significance as it occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals tested.

The variation in motor activity did not indicate a relation with treatment. Despite not reaching statistical significance, the mean high sensor count for females is increased at 1000 mg/kg. This is attributable to the high counts for female no. 74. The very high values for this female may be due to extraneous factors like movement of the tail or head bobbing. If you discount this female’s data for high counts, the mean for Group 4 is 156, and thus, comparable to control values.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.

The significantly kidney to body weight ratios seen for both sexes at 450 mg/kg compared to control animals were considered not to be a sign of toxicity because they remained within the range considered normal for animals of this age and strain and occurred in the absence of a dose-dependent distribution and any microscopic indications of toxicity.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

Incidental findings among control and treated animals included a focus/foci on the lungs, stomach glandular mucosa, and/or preputial glands, reddish or dark red discoloration of the stomach glandular mucosa, mesenteric and mandibular lymph nodes and/or thymus, discoloration of the preputial glands, enlargement of the mandibular lymph node, soft or hard nodule/nodules on the tail of the epididymis, clitoral glands, and/or left uterine horn, uterus contains fluid, diaphragmatic hernia of the liver, and alopecia. The incidence of these findings was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic findings.

No abnormalities were seen in the reproductive organs of the suspected non-fertile animals (Group 2 female 57 and male 17) which could account for their infertility.

All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain.

Staging of spermatogenesis did not provide any evidence of impairment of the spermatogenetic cycle.

Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance related adverse effects were observed.

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Blown rapeseed oil was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 150, 450 and 1000 mg/kg/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 41-45 days).

Parental results:
No parental toxicity was observed at any dose level.

No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

In conclusion, treatment with blown rapeseed oil by oral gavage in male and female Wistar Han rats at dose levels of 150, 450 and 1000 mg/kg body weight/day revealed no parental toxicity up to 1000 mg/kg body weight/day.

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.