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Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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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:
02 March 2010 - 16 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to GLP and OECD guidelines. A reliability of 2 is assigned in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID, due to the read-across purpose.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: not determined
- Fasting period before study: none
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon 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 cages (MIII type; height 15 cm) with sterilised 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 the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-21.4°C
- Humidity (%): 22 -81%
- 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: 02 March 2010 To: 19 April 2010
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 homogenized to a visually acceptable level. Adjustment was made for specific gravity of the test substance and the vehicle.

VEHICLE
On Day 1 of the study, the test substance was administered undiluted. From Day 2 of the study onwards, the test substance changed from being administered undiluted to a formulation because the viscosity of the substance made accurate measurement of the very low dose volumes required for Group 2 too difficult to administer.
- Justification for use and choice of vehicle (if other than water): based on trail formulations performed at NOTOX
- Concentration in vehicle: 0, 75, 225 and 500 mg/mL
- Dose volume:
On Day 1: the dose volume (ml/kg body weight) was calculated as: Dose level (g/kg) / density (g/cm3)
From Day 2 onwards: 2 ml /kg body weight.

Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analyses were conducted, since no analytical method could be developed for formulations of the test substance in corn oil.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 42-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Female 51 (Group 2) was not dosed during littering.
Frequency of treatment:
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.
Remarks:
Doses / Concentrations:
0, 150, 450 and 1000 mg/kg
Basis:

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 492994)
-Results of the dose range finding study are described in End point study record Repeated dose toxicity: oral. NOTOX Project 492993
-For the main study, 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 only
Positive control:
no
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, at least 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.

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.

FOOD CONSUMPTION: Yes
- 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.

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

WATER CONSUMPTION: 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. Several animals were necropsied after approximately 20.5 hours of fasting which was longer than the maximum time of 20 hours allowed for fasting. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.
- How many animals: 5/sex/group (Females: with live offspring only)
- 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, Prothrombin time, Activated Partical thromboplastin time

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. Several animals were necropsied after approximately 20.5 hours of fasting which was longer than the maximum time of 20 hours allowed for fasting. The fasting period was only slightly longer and was considered not to have adversely affected the clinical laboratory, macroscopic or microscopic findings.
- How many animals: 5/sex/group (Females: with live offspring only)
- 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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females (with live offspring) 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 and motor activity test
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 number of former implantation sites and corpora lutea was 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):

Selected 5 animals/sex/group and female no.78 that was killed in extremis (and who had a total litter loss):
Identification marks: not processed, Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Coagulation gland, Duodenum, Epididymides*, (Eyes with optic nerve (if detectable) and Harderian gland)*, 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), (Esophagus), 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*, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions

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

ORGAN WEIGHTS: Yes
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

Selected 5 animals/sex/group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (including cervix), Prostate*, Seminal vesicles including coagulating glands*, Thyroid (including parathyroid)*,
* weighed when fixed for at least 24 hours.

All remaining males:
Epididymides
Testes

HISTOTECHNOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of the selected 5 males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
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.
- The preserved organs and tissues of the animal that was killed in extremis.
- 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

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Inadvertently, the thymus of Animal 41 (Group 1) was not available for histopathology and the skin abnormality from pup 10 from litter 58 (Group 2) was not fixed. These tissues were not discernable at necropsy or trimming (thymus), or were erroneously not collected at necropsy (skin abnormality). Sufficient data was available for an accurate evaluation.
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955; many-to-one t-test) 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 (Miller, 1981; many-to-one rank test) 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 following additional methods of statistical analysis were used:
The number of corpora lutea was transformed by using 1/x to obtain a normal distribution. This was followed by an ANOVA. The Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and 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.
Clinical signs:
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher total bilirubin (both sexes), higher creatinine (males), and higher sodium (males) levels were noted at 1000 mg/kg (Group 4) compared to controls. Means for these values only just exceeded or remained within the range considered normal for rats of
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At 1000 mg/kg, significantly higher liver to body weight ratios were observed for both sexes compared to controls. Means of these changes only just exceeded (females) or remained within (males) the range considered normal for rats of this age and strain.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One female (no. 78) at 1000 mg/kg (Group 4) was euthanized in extremis on Day 2 of the lactation period. At histopathology, severe atrophy of the lympho-hemopoeitic system was determined to be the cause of morbidity for this animal. This was considered to be spontaneous in nature. In the absence of similar findings in any other animal, it was not considered to be treatment related.

CLINICAL SIGNS
No toxicologically relevant clinical signs were noted up to 1000 mg/kg (Group 4).

Hunched posture, moderate lethargy, piloerection and lean appearance were noted for female no. 78 in the days prior to her euthanasia. In the absence of similar findings from any other animals in this dose group, and in the absence of corroborative macroscopic or microscopic findings that were considered to be toxicologically relevant, these findings were not considered to be related to treatment with Standolized linseed oil.

At 450 mg/kg, swelling of the right shoulder was noted over most of the treatment period for one male (no. 24). Because it was only observed for a single male at the mid dose, it was not considered to be related to treatment with Standolized linseed oil. For this animal, a yellowish hard nodule on the esophagus was noted at macroscopic examination and marked granulomatous inflammation adjacent to the esophagus (histological correlate of the nodule) with food fibers found in the inflammation were noted at histopathological examination. While no definitive cause could be determined for these findings, complications with the gavage procedure cannot be excluded.

Incidental findings that were noted included hunched posture (transient for one control and one Group 4 female), rales (transient), salivation, alopecia and/or scabbing of the neck or back. 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 not considered to be signs of toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

Females at 450 mg/kg (Group 3) showed a statistically significant decrease in both absolute and relative food consumption over Days 0-4 of the post-coitum period. Because the change was isolated to a single measurement period, there was no dose response relationship, and values remained within the range of data considered normal for animals of this age and strain, it was not considered to be treatment related.

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with Standolized linseed oil.

The statistically significant increase of prothrombin time (PT) seen for females at 1000 mg/kg (Group 4) was considered not to be toxicologically relevant as the change was slight and 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 nos. 5 (Group 1, control) and 63 (450 mg/kg, Group 3), and the higher white blood cell count also noted for no. 5, were not considered to be toxicologically relevant as they occurred for individual animals in the absence of any dose response relationship. This type of shift in white blood cells was considered to be a secondary non-specific response to stress and not to be treatment related.

The higher reticulocytes and red blood cell distribution width (RDW) seen for female no. 71 (Group 4, 1000 mg/kg) were not considered to be adverse as the values were just slightly outside of the range of data considered to be normal for animals of this age and strain and the remaining animals of this group showed values within the normal range.

CLINICAL CHEMISTRY
Higher total bilirubin (both sexes), higher creatinine (males), and higher sodium (males) levels were noted at 1000 mg/kg (Group 4) compared to controls. Means for these values only just exceeded or remained within the range considered normal for rats of this age and strain.
Higher creatinine and sodium were also noted for males at 450 mg/kg (Group 3).

At 150 mg/kg, the higher glucose values seen for males and the lower alkaline phosphatase (ALP) values seen for females were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. Similarly, the higher alanine aminotransferase (ALAT) value (males; 150 mg/kg) was considered to have arisen as a result of slightly high values for male no. 13.

No explanation can be given for the very high bile acid value seen for male no. 1. There would be no difference between the control and treatment groups even if this value was excluded, and since this was noted for a control animal, it is not indicative of any treatment related toxicity.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS
At 1000 mg/kg, significantly higher liver to body weight ratios were observed for both sexes compared to controls.
Means of these changes only just exceeded (females) or remained within (males) the range considered normal for rats of this age and strain.

At 450 mg/kg statistically significant increases in absolute adrenal weight (males), absolute brain weight (females) and kidney to body weight ratios (males) were seen. A statistically significant reduction in both the absolute thyroid weight and the thyroid to body weight ratio was observed for females at 450 mg/kg and 150 mg/kg. The statistical significance for these parameters may be attributable to relatively low or high values seen in concurrent controls. These changes in organ weights and organ to body weight ratios remained within the range considered normal for this age and strain. In the absence of a dose-dependent distribution and any microscopic indications of toxicity, these changes were not considered to be toxicologically relevant.

Male no. 5 (Group 1, control) had a higher absolute spleen weight, which correlated with his macroscopic finding of an enlarged spleen.

GROSS PATHOLOGY
Necropsy did not reveal any treatment related alterations.

Macroscopic findings noted for the female euthanized in extremis (Group 4; no. 78) included emaciated, accentuated lobular pattern of the liver, enlarged adrenal glands, reduced size of the spleen and thymus, and reddish discoloration of the thymus and the mesenteric lymph node.

There were a few macroscopic findings that were notable but not attributable to treatment with the test item. Male no. 5 (Group 1) had a yellowish hard nodule on the ventricle of the heart and an enlarged spleen. Female no. 45 (Group 1) was noted with the lungs grown together with the diaphragm, an enlarged bronchial lymph node and one adrenal gland reduced in size. Male no. 24 (Group 3) had a yellowish hard nodule on the esophagus. See section 7.2.10 for detail on the microscopic findings noted for these animals. None of the aforementioned findings were attributed to treatment with the test substance.

Incidental findings among control and treated animals included alopecia or scabbing, accentuated lobular pattern of the liver, dark red or reddish focus on the stomach glandular mucosa, tan discoloration or a gray-white hard focus on the clitoral glands, reddish hard nodule on the uterine adipose tissue, yellowish soft focus on the liver, thymus reduced in size, and uterus contains fluid. 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.

HISTOPATHOLOGY
No toxicologically significant findings were noted at histopathological examination up to 1000 mg/kg.

Histopathology findings for Animal 78 (Group 4, 1000 mg/kg) that was euthanized in extremis included marked lymphoid atrophy of the thymus (histologic correlate of reduced in size), moderate submucosal edema and slight mucosal atrophy of the colon, moderate lymphoid atrophy of the spleen (histologic correlate of reduced in size), moderate lymphoid atrophy and slight sinus histiocytosis of the mesenteric lymph node, moderate bilateral vacuolation of tubular epithelium of the kidney, slight multifocal bilateral vacuolation of zona fasciculate of the adrenal glands and marked myeloid atrophy of the sternal bone marrow.
The severe atrophy of the lympho-hemopoeitic system was the cause of morbidity in this animal. No other treated animal had any indication of atrophy of the lympho-hemopoeitic system, and as such, these findings are regarded as incidental and unrelated to treatment.

Microscopic findings of note consisted of marked thrombo-endocarditis of the tricuspidalis valve (histologic correlate of nodule) with hair particles in the centre of the thrombus and slight myeloid hyperplasia in the sternal bone marrow (Animal no. 5; Group 1, control), moderate granulation tissue encapsulating foreign material in the lung (histologic correlate of lungs grown together with the diaphragm), slight lymphoid hyperplasia of the bronchial lymph node (histologic correlate of enlarged) and marked granulomatous inflammation of the largest adrenal gland (Animal no. 45; Group 1, control). Marked granulomatous inflammation adjacent to the esophagus (histologic correlate of nodule) with food fibers found in the inflammation (Animal no. 24; Group 3; 450 mg/kg) was also noted. While no definitive cause could be determined for these effects, the possibility of gavage complications contributing to these findings cannot be excluded.

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

No abnormalities were seen in the reproductive organs of the suspected non-fertile animals (female 49 and male 9 Group 1 (control), female 59 and male no. 19 Group 2 (150 mg/kg) and female no. 74 and male no. 34 of Group 4 (1000 mg/kg)) which could account for their infertility.

Staging of spermatogenesis did not provide any evidence of impairment to the spermatogenetic cycle.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for repeated dose toxicity
Critical effects observed:
not specified
Conclusions:
Standolized linseed 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 42-45 days).
No chemical analyses were conducted, since no analytical method could be developed for formulations of the test substance in corn oil.


Parental results:
No parental toxicity attributable to treatment with Standolized linseed oil was observed at any dose level.

One female at 1000 mg/kg (Group 4) had a total litter loss and was euthanized in extremis on Day 2 of the lactation period. At histopathology, severe atrophy of the lympho-hemopoeitic system was determined to be the cause of morbidity for this animal. This was considered to be spontaneous in nature. In the absence of similar findings in any other animal, it was not considered to be treatment related, and her total litter loss was considered to be secondary to the poor health of the dam.

A number of clinical biochemistry changes were noted at 450 and/or 1000 mg/kg which included higher bilirubin, creatinine and sodium levels in blood. In addition, liver to body weight ratios were increased for both sexes at 1000 mg/kg. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates that would support these changes. Therefore, these changes were considered not to be of toxicological relevance.

Overall, no toxicologically relevant 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 standolized linseed 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, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg/day

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached justification
Reason / purpose:
read-across source
Clinical signs:
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Higher total bilirubin (both sexes), higher creatinine (males), and higher sodium (males) levels were noted at 1000 mg/kg (Group 4) compared to controls. Means for these values only just exceeded or remained within the range considered normal for rats of
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
At 1000 mg/kg, significantly higher liver to body weight ratios were observed for both sexes compared to controls. Means of these changes only just exceeded (females) or remained within (males) the range considered normal for rats of this age and strain.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One female (no. 78) at 1000 mg/kg (Group 4) was euthanized in extremis on Day 2 of the lactation period. At histopathology, severe atrophy of the lympho-hemopoeitic system was determined to be the cause of morbidity for this animal. This was considered to be spontaneous in nature. In the absence of similar findings in any other animal, it was not considered to be treatment related.

CLINICAL SIGNS
No toxicologically relevant clinical signs were noted up to 1000 mg/kg (Group 4).

Hunched posture, moderate lethargy, piloerection and lean appearance were noted for female no. 78 in the days prior to her euthanasia. In the absence of similar findings from any other animals in this dose group, and in the absence of corroborative macroscopic or microscopic findings that were considered to be toxicologically relevant, these findings were not considered to be related to treatment with Standolized linseed oil.

At 450 mg/kg, swelling of the right shoulder was noted over most of the treatment period for one male (no. 24). Because it was only observed for a single male at the mid dose, it was not considered to be related to treatment with Standolized linseed oil. For this animal, a yellowish hard nodule on the esophagus was noted at macroscopic examination and marked granulomatous inflammation adjacent to the esophagus (histological correlate of the nodule) with food fibers found in the inflammation were noted at histopathological examination. While no definitive cause could be determined for these findings, complications with the gavage procedure cannot be excluded.

Incidental findings that were noted included hunched posture (transient for one control and one Group 4 female), rales (transient), salivation, alopecia and/or scabbing of the neck or back. 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 not considered to be signs of toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

Females at 450 mg/kg (Group 3) showed a statistically significant decrease in both absolute and relative food consumption over Days 0-4 of the post-coitum period. Because the change was isolated to a single measurement period, there was no dose response relationship, and values remained within the range of data considered normal for animals of this age and strain, it was not considered to be treatment related.

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with Standolized linseed oil.

The statistically significant increase of prothrombin time (PT) seen for females at 1000 mg/kg (Group 4) was considered not to be toxicologically relevant as the change was slight and 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 nos. 5 (Group 1, control) and 63 (450 mg/kg, Group 3), and the higher white blood cell count also noted for no. 5, were not considered to be toxicologically relevant as they occurred for individual animals in the absence of any dose response relationship. This type of shift in white blood cells was considered to be a secondary non-specific response to stress and not to be treatment related.

The higher reticulocytes and red blood cell distribution width (RDW) seen for female no. 71 (Group 4, 1000 mg/kg) were not considered to be adverse as the values were just slightly outside of the range of data considered to be normal for animals of this age and strain and the remaining animals of this group showed values within the normal range.

CLINICAL CHEMISTRY
Higher total bilirubin (both sexes), higher creatinine (males), and higher sodium (males) levels were noted at 1000 mg/kg (Group 4) compared to controls. Means for these values only just exceeded or remained within the range considered normal for rats of this age and strain.
Higher creatinine and sodium were also noted for males at 450 mg/kg (Group 3).

At 150 mg/kg, the higher glucose values seen for males and the lower alkaline phosphatase (ALP) values seen for females were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. Similarly, the higher alanine aminotransferase (ALAT) value (males; 150 mg/kg) was considered to have arisen as a result of slightly high values for male no. 13.

No explanation can be given for the very high bile acid value seen for male no. 1. There would be no difference between the control and treatment groups even if this value was excluded, and since this was noted for a control animal, it is not indicative of any treatment related toxicity.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.

The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS
At 1000 mg/kg, significantly higher liver to body weight ratios were observed for both sexes compared to controls.
Means of these changes only just exceeded (females) or remained within (males) the range considered normal for rats of this age and strain.

At 450 mg/kg statistically significant increases in absolute adrenal weight (males), absolute brain weight (females) and kidney to body weight ratios (males) were seen. A statistically significant reduction in both the absolute thyroid weight and the thyroid to body weight ratio was observed for females at 450 mg/kg and 150 mg/kg. The statistical significance for these parameters may be attributable to relatively low or high values seen in concurrent controls. These changes in organ weights and organ to body weight ratios remained within the range considered normal for this age and strain. In the absence of a dose-dependent distribution and any microscopic indications of toxicity, these changes were not considered to be toxicologically relevant.

Male no. 5 (Group 1, control) had a higher absolute spleen weight, which correlated with his macroscopic finding of an enlarged spleen.

GROSS PATHOLOGY
Necropsy did not reveal any treatment related alterations.

Macroscopic findings noted for the female euthanized in extremis (Group 4; no. 78) included emaciated, accentuated lobular pattern of the liver, enlarged adrenal glands, reduced size of the spleen and thymus, and reddish discoloration of the thymus and the mesenteric lymph node.

There were a few macroscopic findings that were notable but not attributable to treatment with the test item. Male no. 5 (Group 1) had a yellowish hard nodule on the ventricle of the heart and an enlarged spleen. Female no. 45 (Group 1) was noted with the lungs grown together with the diaphragm, an enlarged bronchial lymph node and one adrenal gland reduced in size. Male no. 24 (Group 3) had a yellowish hard nodule on the esophagus. See section 7.2.10 for detail on the microscopic findings noted for these animals. None of the aforementioned findings were attributed to treatment with the test substance.

Incidental findings among control and treated animals included alopecia or scabbing, accentuated lobular pattern of the liver, dark red or reddish focus on the stomach glandular mucosa, tan discoloration or a gray-white hard focus on the clitoral glands, reddish hard nodule on the uterine adipose tissue, yellowish soft focus on the liver, thymus reduced in size, and uterus contains fluid. 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.

HISTOPATHOLOGY
No toxicologically significant findings were noted at histopathological examination up to 1000 mg/kg.

Histopathology findings for Animal 78 (Group 4, 1000 mg/kg) that was euthanized in extremis included marked lymphoid atrophy of the thymus (histologic correlate of reduced in size), moderate submucosal edema and slight mucosal atrophy of the colon, moderate lymphoid atrophy of the spleen (histologic correlate of reduced in size), moderate lymphoid atrophy and slight sinus histiocytosis of the mesenteric lymph node, moderate bilateral vacuolation of tubular epithelium of the kidney, slight multifocal bilateral vacuolation of zona fasciculate of the adrenal glands and marked myeloid atrophy of the sternal bone marrow.
The severe atrophy of the lympho-hemopoeitic system was the cause of morbidity in this animal. No other treated animal had any indication of atrophy of the lympho-hemopoeitic system, and as such, these findings are regarded as incidental and unrelated to treatment.

Microscopic findings of note consisted of marked thrombo-endocarditis of the tricuspidalis valve (histologic correlate of nodule) with hair particles in the centre of the thrombus and slight myeloid hyperplasia in the sternal bone marrow (Animal no. 5; Group 1, control), moderate granulation tissue encapsulating foreign material in the lung (histologic correlate of lungs grown together with the diaphragm), slight lymphoid hyperplasia of the bronchial lymph node (histologic correlate of enlarged) and marked granulomatous inflammation of the largest adrenal gland (Animal no. 45; Group 1, control). Marked granulomatous inflammation adjacent to the esophagus (histologic correlate of nodule) with food fibers found in the inflammation (Animal no. 24; Group 3; 450 mg/kg) was also noted. While no definitive cause could be determined for these effects, the possibility of gavage complications contributing to these findings cannot be excluded.

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

No abnormalities were seen in the reproductive organs of the suspected non-fertile animals (female 49 and male 9 Group 1 (control), female 59 and male no. 19 Group 2 (150 mg/kg) and female no. 74 and male no. 34 of Group 4 (1000 mg/kg)) which could account for their infertility.

Staging of spermatogenesis did not provide any evidence of impairment to the spermatogenetic cycle.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for repeated dose toxicity
Critical effects observed:
not specified
Conclusions:
Standolized linseed 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 42-45 days).
No chemical analyses were conducted, since no analytical method could be developed for formulations of the test substance in corn oil.


Parental results:
No parental toxicity attributable to treatment with Standolized linseed oil was observed at any dose level.

One female at 1000 mg/kg (Group 4) had a total litter loss and was euthanized in extremis on Day 2 of the lactation period. At histopathology, severe atrophy of the lympho-hemopoeitic system was determined to be the cause of morbidity for this animal. This was considered to be spontaneous in nature. In the absence of similar findings in any other animal, it was not considered to be treatment related, and her total litter loss was considered to be secondary to the poor health of the dam.

A number of clinical biochemistry changes were noted at 450 and/or 1000 mg/kg which included higher bilirubin, creatinine and sodium levels in blood. In addition, liver to body weight ratios were increased for both sexes at 1000 mg/kg. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates that would support these changes. Therefore, these changes were considered not to be of toxicological relevance.

Overall, no toxicologically relevant 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 standolized linseed 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, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg/day

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
1 - Study was performed in accordance with OECD-guidelines and under GLP-conditions

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a combined repeated dose toxicit/reproscreening study, which was performed in accordance with OECD 422 and under GLP-conditions, standolized linseed 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 42-45 days). No chemical analyses were conducted, since no analytical method could be developed for formulations of the test substance in corn oil.

No parental toxicity attributable to treatment with Standolized linseed oil was observed at any dose level. One female at 1000 mg/kg (Group 4) had a total litter loss and was euthanized in extremis on Day 2 of the lactation period. At histopathology, severe atrophy of the lympho-hemopoeitic system was determined to be the cause of morbidity for this animal. This was considered to be spontaneous in nature. In the absence of similar findings in any other animal, it was not considered to be treatment related, and her total litter loss was considered to be secondary to the poor health of the dam. A number of clinical biochemistry changes were noted at 450 and/or 1000 mg/kg which included higher bilirubin, creatinine and sodium levels in blood. In addition, liver to body weight ratios were increased for both sexes at 1000 mg/kg. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates that would support these changes. Therefore, these changes were considered not to be of toxicological relevance. Overall, no toxicologically relevant 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 standolized linseed 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 No Observed Adverse Effect Levels (NOAEL) of at least 1000 mg/kg/day was derived.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One study available which was performed in accordance with OECD-guidelines and under GLP-conditions

Justification for classification or non-classification

Based on Read Across to Standolized linseed oil, Standolized soybean oil does not have to be classified for repeated dose toxicity in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and Annex VI of DSD (67/548/EEC).