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Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Remarks:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 March 2010 - 19 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Constituent 1
Reference substance name:
Linseed oil, oxidized
EC Number:
272-038-8
EC Name:
Linseed oil, oxidized
Cas Number:
68649-95-6
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Oxidation products of seed oil obtained from Linum usitatissimum, Linaceae (linseed)
Details on test material:
- Name of test material (as cited in study report): Blown linseed oil
- Substance type: Industrial chemical
- Physical state: hazy yellow to brown liquid
- Analytical purity: not indicated, treated as 100% pure
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Other:
Specific Gravity / Density: 0.95 g/cm3 (20°C)
pH (1% in water, indicative range): 5.8-5.1 (determined at NOTOX)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on test animals or test system 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: no data
- Fasting period before study: no
- 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): 18.7 - 21.4°C
- Humidity (%): 30 - 91%
Temporary deviations from the minimum and maximum level of relative humidity occurred in the animal room. Laboratory historical data do not indicate an effect of the deviations.
- 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
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: 03 March 2010 To: 19 April 2010

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered undiluted; control animals received the same volume of corn oil as the high dose animals received of blown linseed oil.
- Justification for use: corn oil was used because the test substance is an oil.
- Dose volume: The test substance was administered undiluted and the dose volume (ml/kg body
weight) was calculated as follows: Dose level (g/kg) / spec. gravity or density (g/cm3).
Group 1 animals received the same dose volume as animals of Group 4 (1.05 ml/kg). Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
no
Remarks:
The test substance was administered undiluted so no analytical analysis of the dosages was needed.
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 individually
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 41-47 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 62 (Group 3) and 75 (Group 4) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, at approximately the same time each day, with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study (NOTOX Project 492968)
- Results of the dose range finding study are described in End point study record Repeated dose toxicity: oral.NOTOX Project 492967
- 5 animals/sex/group for the main study were randomly selected at allocation of fucntional observations, clinical pathology, macroscopic examinations (full list), organ weights (full list) and histopathology. Males: the first 5 males per group, Females: with live offspring only.

Examinations

Maternal 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 conducted 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. On Day 22 of treatment, no arena observations were performed for the males. Sufficient information was available from other observations.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

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 AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, but water was provided; All animals were fasted overnight (with a maximum of approximately 22 hours) prior to planned necropsy.
- 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; All animals were fasted overnight (with a maximum of approximately 22 hours) prior to planned necropsy.
- How many animals: 5/sex/group (Females: with live offspring only)
- Parameters checked were: Alanine aminotransferase, Aspartate aminotransferase, Alkaline p
hosphatase, 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

Oestrous cyclicity (parental animals): Not determined.
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

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 were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (
neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):
Selected 5 animals/sex/group and the animal that was found dead (no. 13): dentification 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 the non pregnant female (no. 44)**: 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. Inadvertently, the clitoral gland from animal no. 73 and the pituitary gland from animal no. 35 were not available for histopathology. Reasons for missing these tissues included that they were not discern ble at necropsy or trimming, or were erroneously not collected at necropsy. Sufficient data was available for evaluation.

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 animal no. 13 which died spontaneously
- 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 1: Male no. 4 and female no.44 (non pregnant)
* Reproductive organs included the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Fetal examinations:
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.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (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.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data
(scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistic values.
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 par
turition

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: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included alopecia and transient chromodacryorrhoea. 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 and were not considered to be toxicologically relevant.
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male (no. 13) at 150 mg/kg was found dead after blood sampling, which was considered to be an accidental death. Deaths at blood sampling are considered to be of incidental nature and no relation to treatment was established.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in body weights and body weight gain were noted.
Body weight gain was significantly higher on Day 8 of the pre-mating period for females at 450 and 1000 mg/kg. This was not considered to be an adverse effect, however, since it was transient and weight loss would be more likely to be seen in the case that treatment-related toxicity were evident.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted. Relative food consumption was significantly increased over Days 7-17 of the post coitum period for females at 150 mg/kg. This was not considered to be treatment related as it occurred in the absence of a dose dependent relationship, and an increase in food consumption is not considered to be adverse
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant reduction in reticulocytes and platelets seen for females receiving 150 mg/kg (Group 2) were considered not to represent a change of biological relevance. The significant reduction in platelets was due to low values obtained for animal nos. 54 and 55. Furthermore, values for both platelets and reticulocytes remained within the range considered normal for this age and strain, and occurred in the absence of a dose-dependent relationship.
The increase in neutrophil counts with concurrently reduced lymphocyte counts seen for animal no. 61 (450 mg/kg, Group 3) was not considered to be toxicologically relevant as it occurred for an individual animal in the absence of any dose response relationship. This shift in type of white blood cells was considered to be a secondary non-specific response to stress and not to be treatment related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
The statistically significant reduction in chloride seen for males at 1000 mg/kg was likely attributable to slightly high values for control animals (nos.3 and 5) because the values at 1000 mg/kg remained within the range considered normal for animals of this age and strain, and as such, the reduction is not considered to be toxicologically relevant. At 150 mg/kg, the significant reduction in calcium (males) and the significant increase in aspartate aminotransferase (ASAT; females) were not considered to be treatment related as all values remained within the range considered normal for this age and strain and occurred in the absence of a treatment-related distribution.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals
tested. Males at 1000 mg/kg (Group 4) had lower motor activity counts for both high and low sensors, though this did not reach a level of statistical significance. The data remained within the range of data available for this age and strain, and in the absence of any relevant clinical signs, like lethargy, it was not considered to be toxicologically relevant.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Ovarian weights and ovary to body weight ratios were significantly reduced for females at 1000 mg/kg. At 450 mg/kg, thyroid weights and thyroid to body weight ratios (females) were significantly reduced compared to controls. This was not considered to be toxicologically relevant, however, as the data remained in the range considered normal for this age and strain and occurred in the absence of a treatment related distribution. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.
One animal at 150 mg/kg (no. 13) died during blood sampling; no relationship to treatment with Blown linseed oil could be established. Incidental findings among treated animals included uterus contains fluid, an enlarged renal lymph node, alopecia of various body regions, and an isolated reddish focus on or reddish discoloration of various regions including: the stomach glandular mucosa, lungs, thymus, clitoral glands, and mandibular and/or mesenteric lymph nodes. 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 in cidence trend. These necropsy findings were therefore not considered to be toxicologically relevant.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There was no morphologic evidence of toxicity to the test item Blown linseed oil. There were no treatment-related macroscopic or microscopic findings, nor were there were any morphological findings in the reproductive organs of either sex which could be attributed to the test item.
Furthermore, the assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

Maternal developmental toxicity

Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The gestation index and duration of gestation were similar between controls and all treated groups.
Other effects:
no effects observed
Description (incidence and severity):
REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No toxicologically relevant effects on reproductive parameters were noted.
Mating, fertility and conception index, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Parturition/Maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of the cage debris of pregnant females revealed no signs of abortion or premature birth and no deficiencies in maternal care were observed.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered to have been unaffected by treatment.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Three pups of the control group, one pup at 150 mg/kg, four pups at 450 mg/kg, and one pup at 1000 mg/kg/day were found dead or missing during the first days of lactation. Pups missing from the control, 450 and 1000 mg/kg groups were most likely cannibalized. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Description (incidence and severity):
CLINICAL SIGNS
Incidental clinical symptoms of pups consisted of small size and a missing tail apex. 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.

GROSS PATHOLOGY
The absence of milk in the stomach was an incidental macroscopic finding noted for the control pup that was found dead and cannibalism was noted for the pup found dead at the first litter check at 150 mg/kg. Incidental macroscopic findings among surviving pups included scales on the right front leg, a missing tail apex and a soft nodule on the left side of the lip region. 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 (fetuses)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
No reproduction/developmental toxicity was observed at any dose level.
NOAEL (reproduction) and NOAEL (developmental): >1000 mg/kg/day.
Executive summary:

Blown 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 41-47 days). No chemical analyses were performed since the test substance was administered undiluted. Parental results: No parental toxicity was observed at any dose level. The significant reduction in ovarian weights and ovary to body weight ratios noted for females at 1000 mg/kg were not considered to represent changes of biological significance since there was no corroborative microscopic findings, the data remained within the range considered normal for this age and strain, and all females mated and delivered healthy pups. No toxicologically significant 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, and microscopic examination). Reproductive/Developmental results: No reproduction/developmental toxicity was observed at any dose level. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.