Linseed oil, epoxidized

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 June 1973 to 6 November 1973

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted and reported by BIBRA - while the in life phase pre-dates study guideline adoption and GLP, the report prepared in 1991 has been QA reviewed and is considered reliable. .

Justification for type of information:

Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

Conducted prior to guideline adoption. 19-20 week dosing period with two interim terminations after 2 or 6 weeks

Principles of method if other than guideline:

No specific guidelines are quoted. The study was completed to provide some toxcity data for a material used as a plasticiser and also with food contact applications. The study as completed was broadly in accordance with the requirements of OECD Guideline 408.

Groups of 15 males and 15 female rats were given dietary concentrations of 0, 0.05, 0.5 or 5.0% ELO. A negative control roup was administered linseed oil at 5.0% (non-epoxidised)
Interim groups were terminated after 2 or 6 weeks treatment at 0, 0.05, 0.5 or 5.0% ELO. Each interim group consisted of five males and five females.

GLP compliance:

no

Remarks:

The study was undertaken before GLP implementation. However the report prepared by BIBRA was audited.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Epoxidised Linseed Oil, ELO

IUPAC Name:

Epoxidised Linseed Oil, ELO

Constituent 2

Reference substance name:

Epoxidised Linseed oil

IUPAC Name:

Epoxidised Linseed oil

Details on test material:

- Name of test material (as cited in study report): Epoxidised Linseed Oil (ELO)

Specification for ELO:
Appearance at 25°C: pale yellow oil, possibly containing some winterised solid that clears on warming to 50°C.
Oxirane oxygen %: 8.85% minimum
Iodine value (cgm.I/gm): 5 maximum
Acid value (mgm KOH/gm): 1.0 maximum
Colour (2" cell) yellow: 4.5 maximum
Colour: (2" cell) red: 1.0 maximum
Refractive Index at 25 °C: 1.4750-1.4780
Specific Gravity at 25°C: 1.020-1.035

Specification for LO:
Appearance at 65°C: clear transparent liquid.
Iodine number (Wijs, min): 177
Saponification number: 189-195
Acid number , max: 4.0 maximum
Colour : Not darker than a freshly prepared solution of 1g K2Cr2O7 in 100 ml of pure sulphuric acid (specific gravity 1.84 )
Foots, max %: Heated oil - 1.0; chilled oil - 4.0
Specific Gravity at 15/15°C: 0.931-0.936

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: A Tuck & Son Ltd, Rayleigh, Essex, UK
- Weight at study initiation: 40-60g
- Fasting period before study: rats were not fasted for dietary administration
- Housing: Five to a cage in grid bottom aluminium cages measuring approx. 45x36x16 cm. These were suspended in racks carrying 24 cages above sheets of paper for collection of excreta. The paper was renewed daily.
- Diet : Spratt's Laboratory Animal Diet No. 2 ad libitum
- Water : ad libitum domestic potable water in cage bottles
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): circa 20 °C
- Humidity (%): 45 - 70 %
- Air changes (per hr): no recirculation of filtered 0.5um air to give 15-20 changes of air per hour.
- Light dark cycle: 14 hours light/10 hours dark

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Spratt's Lab Diet No.1 was ground to a powder. ELO (0.05, 0.5, 5 %) or 0.5 or 5% LO were added by weight and thoroughly mixed in a commercial food mixer. The ground diet served as the control. Fresh batches were prepared weekly. Administration was by admixture with the diet which was given ad lib for up to 20 weeks.

No diet analyses were conducted.

Exposure levels were calculated to be:

Linseed oil
% mg/kg bw/day (males) mg/kg bw/day (females)
5.0 3879.7 4200.0

Epoxidised Linseed Oil
0.05 39.3 41.8
0.5 402.0 448.9
5.0 4292.2 4609.0

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No analyses were carried out to verify the concentration of ELO or LO.

Duration of treatment / exposure:

Administration was by admixture with the diet which was given ad libitum for up to 20 weeks. Interim sacrifice groups were dosed for 2 or 6 weeks, the main study males for 19 weeks and main study females for 20 weeks.

Frequency of treatment:

Test item was provided via diet so the diet was accessible all day. Fresh diet prepared at weekly intervals and new diet suppled daily to each cage.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 %
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.05 %
Basis:
nominal in diet

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0.5 %
Basis:
nominal in diet

Doses / concentrations 4

Remarks:

Doses / Concentrations:
5 %
Basis:
nominal in diet

No. of animals per sex per dose:

15 per sex per group in main study. Five per sex/group in each of two interim termination sub groups

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The results of a structural analogue ESBO (see long term repeatdose study with ESBO also prepared by BIBRA)
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: 2-week and 6 week interim sacrifice groups equate to sub acute exposure scenarios to obtain additional information from the study.
Very few investigations were available at the time the study was conducted. In vivo data were in short supply. In vitro results indicated ELO was of low toxicity to HeLa cells, there was no evidence of mutagenicity from an Ames test with several bacterial strains with or without metabolic activation. The remaining information came from the structuarla analogue ESBO. Consequently most ofthe remaining data in this dossier is obtained by read-across to ESBO.

Positive control:

No details supplied

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Rats were observed regularly but no records of conditional or behavioural changes were maintained.

BODY WEIGHT: Yes
These were recorded on the first day of treatment (day 0), twice per week for the first two weeks and then at weekly intervals to termination at week 6, 19 or 20.

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
Both food and water were measured over the 24 hour period prior to each bodyweight determination.

HAEMATOLOGY: Yes
Blood was taken from the dorsal aorta at termination (both interim sacrifices and final termination). The blood was examined for haemaglobin content, packed cell volume and erythrocyte and total and differential leucocyte counts plus reticulocyte counts.

URINALYSIS: Yes
Urine samples were collected from 12 rats of each sex from the main study during week 12-13 (males) or week 14-15 (females). Urine samples were collected from all rats in the interim sacrifice groups - in week 2 and 5.
The samples were obtained by placing the animals individually in metabolism cages without food or water. The conditions of the collection and examinations were:
- Collected over a 6 hr period following the normal overnight feeding and drinking and examined for volume, concentration (by refractive index), pH and the presence of glucose, blood, bile salts, ketones and proteins
- Collected over a 2 hour period immediately following an oral water load of 25 ml/kg and examined for volume, concentration and content of cells.
- Collected over a 4 hour period commencing 16 hour after an oral water load of 25 ml/kg. Food but not water was available to the animals during the 16 hour period. These samples were examined for volume and concentration.

Sacrifice and pathology:

After 2, 6, 19 or 20 weeks all surviving rats were killed, subject to post-mortem examination with recording of organ weights and samples of tissues preserved. The tissues from all rats were processed for microscopic examination.
ORGAN WEIGHTS: Yes
adrenals, brain, caecum, gonads, heart, kidneys, liver, pituitary gland, small intestine, spleen, stomach, thyroid gland

GROSS PATHOLOGY: Yes
Rats surviving to the end of the study were weighed and the weights of brain, heart, liver, spleen, kidneys, stomach, small intestine, caecum (with and without contents), gonads, pituitary and thyroid recorded. When possible the following tissues, together with any other abnormal tissue, were preserved in 10 % buffered formalin: adrenal glands, aorta, bladder (urinary), brain, caecum, colon, eye, gonads, harderian gland, heart, kidneys, liver, lungs, lymph nodes, mammary gland, muscle (skeletal), nerve (sciatic), oesophagus, pancreas, pituitary gland, prostate, salivary glands, seminal besicles, skin, small intestine, spinal cord, spleen, stomach, thymus gland, thyroid gland, trachea, uterus.

HISTOPATHOLOGY: Yes
All tissues collected were processed for embedding in paraffin-wax, sectioned at approx. 5 um and stained with haematoxylin and eosin.
Additional liver sections were stained with Oil Red O as a marker of intracellular lipid deposition.

Other examinations:

No details supplied

Statistics:

The results of the ELO and LO groups were initially compared statistically with the control group. Then the treated ELO groups were analysed independently with the control.

Comparisons were made with two sided least signifiant, t-test or sample t-test with Satterthwaite's correction or by Mann-Whitney U test. The tests were selected on degree of normality or homoscedasticity as assessed by Kolmogorov-Smirnov tests and Bartlett's test.
Statistical tests were applied to bodyweights, food and water consumption, haematology, urinalysis, organ weights and relative organ weights and histopathological findings.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No unscheduled mortalities. No records maintained for other observations

Mortality:

mortality observed, treatment-related

Description (incidence):

No unscheduled mortalities. No records maintained for other observations

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight changes at the high dose level were not indicative of treatment related effects

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

On several occasion during treatment period food consumption by all ELO groups ws reduced in comparison with basal diet controls. Generally consumption was similar for ELO and LO groups.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

ELO treated rats tended to drink less than base diet controls but water consumption was similar for ELO and LO treated groups

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically significant changes noted

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Minor changes oserved but with no adverse toxicological effects.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver and kidney weights tended to increase in high dose group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related macroscopic observations

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Increased hepatocyte vacuolation, attributed to high lipid content of diet containg 5% ELO

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities for any of the ELO, LO or control groups in the main study. The interim sacrifice groups, killed after 2 or 6 weeks of treatment also had no unscheduled deaths.
No record of clinical observations was retained in the study data and the report gives no details of any clinical signs of reaction to treatment.

BODY WEIGHT AND WEIGHT GAIN
Minor fluctuations in bodyweight were noted for males and females in each of the treatment groups in comparison with basal diet controls but no notable differences were apparent when weights were compared with linseed oil treated controls. Over the course of the 20 weeks of the main study, bodyweights were significantly lower during the fist half of the study for males and females dosed at 5% ELO . In the second half of the study the weights were slightly increased such that by termination there were no significant differences between ELO or LO treated rats and the diet controls.
ELO had no notable adverse effect on bodyweight or weight gain

FOOD CONSUMPTION AND COMPOUND INTAKE
Food and water consumption showed minor variations at various points of recording. Generallya slight reduction in water consumption was commonly recorded forthe ELO treated groups but no clear dose related effects were apparent. Food and water consumption were compared, for ELO groups, with both LO and basal diet controls and the slight significant differences showed no consistent trends to indicate any adverse effects.

HAEMATOLOGY
There were few statistically significant differences between ELO treated and control groups for any of the haematology parameters.
Blood samples were collected after 2, 6 or 19/20 weeks.
After two weeks, reticulocyte counts were reduced for the high dose males and females and intermediate group males. No differences between ELO and LO groups. (There was only one male control value for reticulocytes).
After 6 weeks, the high dose males had a significantly increased lymphocyte count, but a similar increase occurred for LO treated rats.
At study conclusion there were no significant differences for any parametrs for the treated females. The high dose males had a significantly lower eosinophil count.
None of the haematology parameters showed a consistent pattern indicative of a treatment or dose related effect.


URINALYSIS
Renal dilution tests were conducted after 2, 6 or 19 weeks of treatment. The significant results in the high doe males were not replicated among the females and my ahave been a consequence of the larger urine volumes poduced by the males (despite generally lower water consumption values).

Semi-quantitative analyses of the 6-h urine samples showed no toxicologicallysignificant changes for ELO or LO treated rats.


ORGAN WEIGHTS
2-week interim sacrifice
The absolute and relative liver weights were significantly increased for males dosed at 5% ELO (and for the 5% LO treated rats).
The absolute liver weights for the 5% ELO treated females were slightly but not significantly increased, while the relative weight increase was significant.
The relative kidney weights for the high dose group males were increased in comparison with controls and 5% LO treated rats.
Absolute spleen weights were reduced for the high dose males and females. Pituitary weights in the high dose group were also slightly reduced.
Empty caecum weights relative to bodyweight were increased for the high dose females.
Apart from the liver and kidney changes in the high dose group, none of these changes were considered toxicologically notable.

6 week interim sacrifice
Absolute stomach weights were slightly lower for the high dose males. The absolute and relative stomach weights were also significantly lower for males treated at 0.5% ELO. No effects were noted for females.
Relative liver weights were significantly increased for males and females dosed at 5% ELO.
Absolute pituitary gland weights were significantly lower for the high dose males. but relative weights were unaffected.
Relative kidney weights for male and female rats treated at 5% ELO were significantly increased in comparison with controls.
Minor changes were noted for caecum, stomach, adrenal weights in some groups but no dose-relationship.

Terminal sacrifice
The absolute and relative liver and kidney weights for males and females treated at 5% were statistically significantly greater than the controls and the 5% LO treated rats. The percentage increase was 16-30% for both sexes.

Other changes were noted - similar to the 2 and 6 week termination groups - affecting stomach, caecum, pituitary gland, showing slight or significant differences from controls but showing no consistent trends in relation to dose or treatment.


GROSS PATHOLOGY
No treatment-related changes were observed during necropsy.

HISTOPATHOLOGY: NEOPLASTIC
Thickening of the alveolar wall was evident in ELO-treated male and female rats dosed at 5%. No macroscopic changes in the lungs were noted.

Vacuolation of hepatocytes was commonly observed in both control and treated rats. The degree of vacuolation was assigned from examination of Oil Red O stained sections. The degree of vacuolation was greater for the high dose males and females and associated with the presence of lipid.

A higher incidence of tracheal mucosa inflammatory cell infiltrate was apparent for the low dose females - 0.05%

Effect levels

1

Dose descriptor:

NOEL

Effect level:

0.5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Please see results

2

Dose descriptor:

NOAEL

Effect level:

5 other: %

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Minor changes were noted at the highest dose level - 5% ELO in diet, equivalent to more than 4200 mg/kg bw/day. None of the effects were considered toxicologically significant

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

calculated intakes:

Exposure levels were calculated to be:

Linseed oil

% mg/kg bw/day (males) mg/kg bw/day (females)

5.0 3879.7 4200.0

Epoxidised Linseed Oil

0.05 39.3 41.8

0.5 402.0 448.9

5.0 4292.2 4609.0

Applicant's summary and conclusion

Conclusions:

5% ELO in diet administered to rats for up to 20 weeks, resulted in no adverse toxicologically significant effects. A number of slight variaions occurred involving liver and kidney weights, reduced water consumption and hepatocyte vacuolation which were attributed to treatment with ELO. Consequently the NOAEL is considered to be 5% or greater than 4200 mg/kg bw/day but the absolute n effect level was set at the next lower dose level - 0.5% ELO or 402 mg/kg bw/day.
Comparison of ELO with the structual analogue epoxidised soybean oil suggests that the NOAEL of 5% is a good representation of the low toxicity and innocuous nature of the epoxidised oil group.

Executive summary:

Groups of 15 male and 15 female rats of a Wister strain were fed diet containing 0.05, 0.5 or 5 % Epoxidised Linseed Oil (ELO) for 19 -20 weeks. Similar groups were given basal diet to serve as a control or 0.5 or 5.0% Linseed Oil (LO). In addition two interim sacrifice points were scheduled after 2 or 6 weeks of treatment (five male and five female rats were dosed at 0.05, 0.5 or 5% ELO for each sacrifice point).

Bodyweights were recorded at regular ntervals through the study. Food and water intakes were also measured regularly. Terminal blood samples were collected from the dorsal aorta, after 2, 6 or 19/20 weeks. Standard haematology and urinalysis parameters were measured. Various organs were weighed at termination and preserved tissues were examined microscopically.

Initially the bodyweights for the high dose ELO group were reduced in comparison with linseed oil and untreated diet controls, but weight gains were similar for all groups by the end of the treatment period.

Food and water consumption were similar for all treated and control groups.

There were no changes in any of the haematology or urinalysis parameters indicative of treatment-related toxicity.

Increased relative liver weights were evident in the males and females dosed at 5% ELO, in the two interim sacrifices and main study sacrifice. Relative kidney weights in this dose group were also increased for both sexes.

There were no histopathological changes to account for the renal weight increase. The high dose livers showed a greater degree of vacuolated lipid filled hepatocytes. These fatty changes were considered to account for the liver weight increase noted macroscopically but were considered attributable to a higher lipid content of the diet (5% ELO) rather than to any toxic effect of Epoxidised Linseed Oil.

None of the effects observed at 5% ELO were considered to be toxicologically significant. The no observed effect level or NOEL was set at the next lower dose (0.5% ELO) equivalent to 402 mg/kg bw/day. The NOAEL was 5% ELO or 4292 mg/kg bw/day.

For comparison, it was concluded that ESBO (epoxidised soybean oil) following a two year dietary administration, was not carcinogenic when fed to rats at up to 2.5 % of the diet. Further it was concluded that the NOEL was 2.5 % providing an average daily intake of approximately 1000 mg/kg in the males and 1400 mg/kg in the females. 

Much of the data generated for ESBO is used via read-across for ELO. The results of these two sub-chronic or chronic exposure studies confirm that ELO is unlikely to prove more toxic than ESBO and so the data is conservative for read-across for epoxidised linseed oil.