Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels
Type of information:
experimental study
Adequacy of study:
key study
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.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on generations indicated in Effect levels
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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.
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
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of living pups at first litter check was unaffected by treatment. The statistically significant reduction in the viability index observed for females at 1000 mg/kg was attributable to the total litter loss of female no. 78. Her total litter los
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lactation, and body weights of pups at 450 mg/kg were significantly lower than controls on Day 4 of lactation. This was not secondary to reduced maternal body weights, however, as maternal weights remained within the range of controls over the treatment p
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The statistically significant reduction in the viability index observed for females at 1000 mg/kg was attributable to the total litter loss of female no. 78. Her total litter loss was considered to be due to the poor health of the dam and was not related to treatment with the test substance.

No pups of the control group or of the 150 and 450 mg/kg groups were found dead at first litter check. At 1000 mg/kg, the number of dead pups was significantly higher than controls with 12 pups found dead at first litter check. Female no. 78 had 9 of the 12 pups at 1000 mg/kg that were found dead, and the other 3 pups came from three additional litters. Excluding female no. 78 from consideration, the number of dead pups per litter was not significantly higher than historical control data. However, incidence of dead pups at first litter check, i.e. the number of litters with dead pups, was higher than normally seen. As such, a test substance effect cannot be excluded.
Similarly, there were no postnatal losses in the control or 150 mg/kg groups, while there were 1 and 5 pups that either died or were missing in the 450 and 1000 mg/kg groups during the first days of lactation, respectively. Female no. 78 had 4 of the 5 pups found dead on Day 2 of lactation, and after removing her litter from consideration, there was no indication of a treatment related increase in postnatal loss.
Death of all pups of Female no. 78 was considered due to bad health of this female.
Missing pups were most likely cannibalized.


CLINICAL SIGNS (OFFSPRING)
No milk in the stomach, small size and cold appearance were noted at the first litter check for the four pups that died spontaneously on Day 2 for female no. 78.
Incidental clinical symptoms of pups consisted of small size, blue spot on the nose or head, scabbing of the neck or snout and/or a wound on the abdomen. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
Body weights of pups at 1000 mg/kg were significantly lower than controls over Days 1 and 4 of lactation, and body weights of pups at 450 mg/kg were significantly lower than controls on Day 4 of lactation. This was not secondary to reduced maternal body weights, however, as maternal weights remained within the range of controls over the treatment period and the reduced body weight of pups was consistent over all litters (excluding no. 78) at 450 and 1000 mg/kg.

GROSS PATHOLOGY (OFFSPRING)
No milk was noted for all of female no. 78’s pups that were dead at the first litter check or that died spontaneously on Day 2 of lactation.
Incidental macroscopic findings of pups that were found dead at the first litter check included autolysis and the absence of milk in the stomach. Incidental macroscopic findings among surviving pups included scabs on the neck and a 3 x 3 mm blue focus on the bones of the head. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.


Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for Reproduction toxicology
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for Developmental toxicity
Reproductive 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).


Reproductive/Developmental results:
No reproductive toxicity was noted up to 1000 mg/kg. Developmental toxicity was noted that consisted of an increased number of litters with dead pups at first litter check at 1000 mg/kg and reduced body weights for pups over Days 1 and/or 4 at 1000 and 450 mg/kg, respectively.


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. Developmental toxicity was seen, characterized by an increased number of litters with dead pups at 1000 mg/kg, and reduced body weights of the pups at 450 and 1000 mg/kg. No reproduction toxicity was observed up to 1000 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg/day
Reproduction NOAEL: at least 1000 mg/kg/day
Developmental NOAEL: 150 mg/kg/day

Data source

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

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Standolized linseed oil
- Substance type: Industrial chemical
- Physical state: Hazy yellow/brown viscous liquid
- Analytical purity: Not indicated by the sponsor; treated as 100% pure
- Lot/batch No.: 09.317.013
- Expiration date of the lot/batch:21 December 2010 (allocated by NOTOX, 1 year after receipt of the test substance)
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Other:
Density: 0.97 g/cm3 (20°C)
pH (1% in water, indicative range): 7.7-6.6 (determined at NOTOX)

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After successful mating each pregnant female was caged (how): individually
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.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 13 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 450 and 1000 mg/kg
Basis:

No. of animals per sex per dose:
10 animals/sex/group + 463 pups
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

Examinations

Parental animals: Observations and examinations:
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:

Group Female/male nos. Reason
1 49/9 Female appeared to be non-pregnant
2 59/19 Female appeared to be non-pregnant
3 -- --
4 74/34 Female appeared to be non-pregnant

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.

Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
Parameters examined in all male parental animals:
testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine the staging of spermatogenesis

OTHER:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Litter observations:
PARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

Mortality / Viability:
The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated

Clinical signs:
At least once daily, detailed clinical observations were made in all animals.

Body weights:
Live pups were weighed on Days 1 and 4 of lactation.

Sex:
Sex was determined for all pups on Days 1 and 4 of lactation.


GROSS EXAMINATION OF DEAD PUPS: Yes
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.
Postmortem examinations (parental animals):
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:

Group Female/male nos. Reason
1 49/9 Female appeared to be non-pregnant
2 59/19 Female appeared to be non-pregnant
3 -- --
4 74/34 Female appeared to be non-pregnant

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.
Postmortem examinations (offspring):
SACRIFICE
-Pups surviving to planned termination were killed by decapitation on lactation Days 5-7.

GROSS NECROPSY
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin for possible further examination.

HISTOPATHOLOGY / ORGAN WEIGHTS: No

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.
Reproductive indices:
For each group, the following calculations were performed:

Mating (%): Number of females mated/Number of females paired x 100

Fertility index (%): Number of pregnant females/Number of females paired x 100

Conception index (%): Number of pregnant females/Number of females mated x 100

Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100

Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100

Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of living pups at first litter check was unaffected by treatment. The statistically significant reduction in the viability index observed for females at 1000 mg/kg was attributable to the total litter loss of female no. 78. Her total litter los
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
lactation, and body weights of pups at 450 mg/kg were significantly lower than controls on Day 4 of lactation. This was not secondary to reduced maternal body weights, however, as maternal weights remained within the range of controls over the treatment p
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
The statistically significant reduction in the viability index observed for females at 1000 mg/kg was attributable to the total litter loss of female no. 78. Her total litter loss was considered to be due to the poor health of the dam and was not related to treatment with the test substance.

No pups of the control group or of the 150 and 450 mg/kg groups were found dead at first litter check. At 1000 mg/kg, the number of dead pups was significantly higher than controls with 12 pups found dead at first litter check. Female no. 78 had 9 of the 12 pups at 1000 mg/kg that were found dead, and the other 3 pups came from three additional litters. Excluding female no. 78 from consideration, the number of dead pups per litter was not significantly higher than historical control data. However, incidence of dead pups at first litter check, i.e. the number of litters with dead pups, was higher than normally seen. As such, a test substance effect cannot be excluded.
Similarly, there were no postnatal losses in the control or 150 mg/kg groups, while there were 1 and 5 pups that either died or were missing in the 450 and 1000 mg/kg groups during the first days of lactation, respectively. Female no. 78 had 4 of the 5 pups found dead on Day 2 of lactation, and after removing her litter from consideration, there was no indication of a treatment related increase in postnatal loss.
Death of all pups of Female no. 78 was considered due to bad health of this female.
Missing pups were most likely cannibalized.


CLINICAL SIGNS (OFFSPRING)
No milk in the stomach, small size and cold appearance were noted at the first litter check for the four pups that died spontaneously on Day 2 for female no. 78.
Incidental clinical symptoms of pups consisted of small size, blue spot on the nose or head, scabbing of the neck or snout and/or a wound on the abdomen. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
Body weights of pups at 1000 mg/kg were significantly lower than controls over Days 1 and 4 of lactation, and body weights of pups at 450 mg/kg were significantly lower than controls on Day 4 of lactation. This was not secondary to reduced maternal body weights, however, as maternal weights remained within the range of controls over the treatment period and the reduced body weight of pups was consistent over all litters (excluding no. 78) at 450 and 1000 mg/kg.

GROSS PATHOLOGY (OFFSPRING)
No milk was noted for all of female no. 78’s pups that were dead at the first litter check or that died spontaneously on Day 2 of lactation.
Incidental macroscopic findings of pups that were found dead at the first litter check included autolysis and the absence of milk in the stomach. Incidental macroscopic findings among surviving pups included scabs on the neck and a 3 x 3 mm blue focus on the bones of the head. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.


Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for Reproduction toxicology
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: NOAEL for Developmental toxicity

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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).


Reproductive/Developmental results:
No reproductive toxicity was noted up to 1000 mg/kg. Developmental toxicity was noted that consisted of an increased number of litters with dead pups at first litter check at 1000 mg/kg and reduced body weights for pups over Days 1 and/or 4 at 1000 and 450 mg/kg, respectively.


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. Developmental toxicity was seen, characterized by an increased number of litters with dead pups at 1000 mg/kg, and reduced body weights of the pups at 450 and 1000 mg/kg. No reproduction toxicity was observed up to 1000 mg/kg/day.

Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg/day
Reproduction NOAEL: at least 1000 mg/kg/day
Developmental NOAEL: 150 mg/kg/day