Linseed oil, oxidized

Endpoint summary

Description of key information

OECD 422: no evidence of repeated dose toxicity (NOAEL: >1000 mg/kg bw/day)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 March 2010 - 19 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to GLP and OECD guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

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

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Remarks:

Control animals received corn oil with the same volume the high dose animals received.

Details on oral 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

Details on analytical verification of doses or concentrations:

The test substance was administered undiluted so no analytical analysis of the dosages was needed.

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.

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

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

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early morning/late afternoon).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were 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; approximately 2 hours over the maximum time of 20 hours fasting allotted. The fasting period was only slightly longer and was considered not to have adversely affected the integrity of the study) 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; approximately 2 hours over the maximum time of 20 hours fasting allotted. The fasting period was only slightly longer and was considered not to have adversely affected the integrity of the study) prior to planned necropsy.
- 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 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):
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 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 discernable 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.

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One 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.


CLINICAL SIGNS
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.


BODY WEIGHT AND WEIGHT GAIN
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 FOOD EFFICIENCY
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.


HAEMATOLOGY
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 CHEMISTRY
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.

NEUROBEHAVIOUR
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 WEIGHTS
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 PATHOLOGY
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 incidence trend. These necropsy findings were therefore not considered to be toxicologically relevant.


HISTOPATHOLOGY
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.

Key result

Dose descriptor:

NOAEL

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

Key result

Critical effects observed:

no

Conclusions:

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

Reference 2

Endpoint:

sub-chronic toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because the substance is unreactive, insoluble and not inhalable and there is no evidence of absorption and no evidence of toxicity in a 28-day 'limit test' and human exposure is limited

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Justification for classification or non-classification

Based on the information available, the substance does not have to be classified for repeated dose toxicity according to EU CLP (EC 1272/2008 and its amendments).