Soybean oil, epoxidized, ether with ethylene glycol

Endpoint summary

Description of key information

A modern guideline-compliant screening oral study with the read-across substance ETP is available.  Older one-year dietary studies in the rat and dog with the read-across substance ESBO are also available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral, other

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

November 24, 2004 - June 13, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study completed in accordance with current guidelines - OECD 422.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see 'Overall Remarks'

GLP compliance:

yes

Species:

rat

Strain:

other: HanBrl:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd. Switzerland
- Age at study initiation: 10 weeks minimum
- Weight at study initiation: Males - 291-342 g Females - 180-223g
- Fasting period before study: Only fasted before collection of blood samples
- Housing: Animals were housed in Makrolon cages with wire mesh tops and standard granulated softwood bedding. During the prepairing period, males and females were housed individually. Cages of males were interspersed amongst those holding females to promote the development of regular estrous cycles. During the pairing period; rats were housed one male/one female in Makrolon pairing cages. After mating or at the end of the pairing period, the males and the females were housed individually again. During the lactation period (until day 4 of lactation), dams were housed together with their litters.
- Diet ( ad libitum): Pelleted standard Kliba-nafag 3433 rat/mouse maintenance diet (Batch no. 53/04)
- Water ( ad libitum): Fullinsdorf community potable tap water provided in cages bottles
- Acclimation period: Minimum of 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 5 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark and at least 8 hours light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and approximately 80 % of the vehicle added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. The remaining vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration at weekly intervals. During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): ETP is stable in Corn Oil. Analytical evaluations were conducted by RCC.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for determination of concentration, homogeneity and stability (7 days) of the dose formulations were taken immediately after the first dose preparation. Determination of concentration and homogeneity of these samples was performed prior to the first dosing occasion. Additionally, samples for determination of concentration and homogeneity were taken once during gestation.
In addition to these two sets of samples,.samples were also retained from each set of weekly dose formulations (three samples from top, middle and bottom). These samples were not analysed since no discrepancy (outside the specified range) was noted in those samples scheduled for analysis.
For determination of concentration and homogeneity three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. Stability samples (approximately 2 g) were taken from the middle only. The samples were frozen (-25 °C to -15 °C) pending analysis. Analysis was performed using GC.
Actual sample concentrations had to be within the accepted limit of 80 % - 120 % of the nominal concentration. Results of homogeneity must not deviate more than 15 % from the respective mean value. Stability results were accepted if the deviation from mean homogeneity results was not higher than 10 %. After analysis and evaluation of the results, the dose formulation samples were discarded at the discretion of the principle investigator for the analytical phase.

Duration of treatment / exposure:

After acclimatization, animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to Day 4 of lactation (4 or 5 days post-partum). Males were dosed for at least 28 days and until the day prior to scheduled necropsy. Males were terminated on day 29 and pups on day 4 post-partum. The dams were terminated on day 5 or 6 post-partum

Frequency of treatment:

ETP was administered once daily orally (by gavage). All animals received a dose volume of 2 mL/kg

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

40 males, 10 per group
40 females, 10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

No details supplied
- Dose selection rationale: The purpose of this study was to generate preliminary information concerning the effects of Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, it
provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. ETP was administered once daily orally (by gavage) to males for at least 28 days and to female rats throughout the prepairing and pairing periods until day 3 of lactation. The choice of 100, 300 and 1000 mg/kg bw/day is a standard selection for a dose range-finding investigation.

- Rationale for animal assignment (if not random): P generation allocated randomly
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable

Positive control:

No positive control included in the study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All animals were checked at least twice daily for any mortalities. All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health . Additionally, the females were observed for signs of difficult or prolonged parturition.

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the first administration and then at weekly intervals thereafter, a detailed clinical observation was performed outside the home cage. Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and automatic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern); changes in gait, posture and response to handling as well as the presence of clonic and tonic movements, stereotypies or bizarre behaviour.

At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters from a modified Irwin screen test were performed for five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following: (a) Cage side observations: unusual body movements, abnormal behaviour, (b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation (c) Open field observations: level of ambulatory activity including rearing, responsiveness to sharp noise (d) Categorical observations: hair coat, behaviour, respiration, muscle movements (e) Measurements/Counts: hindlimb/forelimb grip

BODY WEIGHT: Yes
The animals were weighed daily throughout the entire study.

FOOD CONSUMPTION AND COMPOUND INTAKE :
Males: Food consumption was recorded weekly during the pre-pairing and post-pairing period.
Females: Food consumption was recorded for the following periods: days 1-8 and 8-14 of the pre-pairing period; days 0-7, 7-14 and 14-21 post coitum and days 0-4 post partum. Food consumption was not recorded during the pairing period since these would have been mixed values of males and females.

HAEMATOLOGY: Yes
Blood samples were obtained on the day of scheduled necropsy from all P generation males after they had been fasted overnight. Blood samples of P generation females were obtained on day 5 or 6 post partum after the females had been fasted overnight. Blood samples were collected from the retro orbital sinus with the animals under light isoflurane anaesthesia.
Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. The following haematology parameters were determined: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, platelet count, total leukocyte count, differential leukocyte count, coagulation, thromboplastin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined: glucose, urea, creatinine, bilirubin (total), cholesterol (total), Aspartate aminotransferase, Alanine aminotransferase, bile acids, alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, protein (total), albumin, globulin, albumin/globulin ratio.

LACTATION AND LITTER DATA:
Day 0 of lactation was the day on which a female had delivered all her pups. The litters were examined for litter size, live birth, stillbirth, and any gross anomalies. The sex ratio of the pups was recorded. The dams were caged together with their litters until day 4 of lactation. Pups were weighed individually (without identification) on days 0 (if possible), and with identification on days 1 and 4 post partum. The dams and pups were observed daily for survival and behavioural abnormalities in nesting and nursing.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Males were sacrificed on day 29 after treatment for 28 days. Pups were sacrificed on day 4 post partum. Females were sacrificed on day 5 or 6 post partum. The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. Dead pups (except where excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.

Of all parental animals the following tissues were preserved in neutral phosphate buffered 4 % formaldehyde solution: gross lesions, prostate, testes (in Bouin’s fixative), seminal vesicles with coagulation gland, ovaries, epididymides (in Bouin’s fixative), brain, spinal cord, small and large intestines (incl. Peyer’s patches), stomach, liver, kidneys, adrenals, spleen, heart, uterus with vagina, thymus, trachea and lungs (preserved by inflation with fixative and then immersion), thyroid, lymph nodes (mesenteric and mandibular), urinary bladder, peripheral nerve, and bone marrow.

HISTOPATHOLOGY: Yes
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). For liver and thyroid examinations were extended to the animals of the other dosage groups, since treatment-related changes were seen in the highest dose group. All gross lesions were examined.

ORGAN WEIGHTS: Yes
For all adult female and male animals in each group the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken: liver, brain, adrenals, thymus, kidneys, spleen, heart, testes and epididymides.

Other examinations:

Deailed above

Statistics:

Dunnett t-test, Steel (rank) test, Fischer's Exact test

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:

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

During necropsy of parent animals no treatment-related findings were noted. For males treated at 300 or 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

inimal hepatocellular hypertrophy in animals treated at 1000 mg/kg bw/d

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortalities were observed. No treatment-related effects were seen. Neither food consumption nor body weight development were affected at any dose level.

BODY WEIGHT AND WEIGHT GAIN
No effects seen in males or females at any dose level

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was slightly higher in the male group 4. This was incidental and of no toxicological relevance.

HAEMATOLOGY
No effects seen

CLINICAL CHEMISTRY
No effects seen

NEUROBEHAVIOUR
Locomotor activity was statistically higher in group 3 and 4 males than in the respective control. This was attributable to normal biological variation.

ORGAN WEIGHTS
During necropsy of parent animals no test item-treated findings were noted. For males treated at 300 or 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased.

GROSS PATHOLOGY
The intergroup incidence of various spontaneous changes gave no indication of an effect of treatment. No treatment related macroscopic abnormalities were noted.

HISTOPATHOLOGY:
Liver- Minimal hepatocellular hypertrophy in animals treated at 1000 mg/kg bw/day. This change was considered to represent an adaptive reaction most likely induced by an increased biotransformation of the test item. Therefore, it was not assessed as an adverse effect.
Thyroid- Increased incidence of minimal follicular cell hypertrophy in animals treated at 1000 mg/kg bw/day. No test item- related histopathological findings were noted in the reproductive organs of either sex. In particular, the assessment of the integrity of the spermatogenetic cycle did not reveal any evidence of impaired spermatogenesis.

REPRODUCTION DATA
Fertility rate was high resulting in at least 9 litters per group for evaluation of reproduction data. There were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to scheduled pup sacrifice on Day 4 post partum at any dosage.
Litter Data:
No test item-related abnormal findings were noted for pups at first litter check or during the first 4 days post partum. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.
Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. Mean pup weight development during the first 4 days post partum was unaffected by treatment with the test item.
F1 Pups Necropsy findings:
No test item-related macroscopic findings were noted during necropsy of F1 pups.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The only effect noted at this concentration was an increased liver weight

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Higher doses resulted in increased liver weight

Critical effects observed:

not specified

Table 2: F0 animals breeding for F1 litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
# of females paired	10	10	10	10
# of females mated	10	10	10	10
# of pregnant females	10	10	10	10
# of females delivering pups	10	10	10	10
# of females with live pups on day 4 post partum	10	9 (A)	10	10

A = For female 52 only two dead pups were noted at first litter check

Table 3: Organ Weights (Gram)

		Group 1 0 mg/kg	Group 2 100 mg/kg	Group 3 300 mg/kg	Group 4 1000 mg/kg
Liver Males	Mean	8.33	8.73	9.38*	9.71*
	St. Dev.	0.78	1.00	0.81	0.56
	N	10	10	10	10
Liver Females	Mean	8.02	9.03	8.40	9.43*
	St. Dev.	0.81	1.42	1.46	0.98
	N	10	10	10	9

 

Conclusions:

Effects in this study were limited to increased liver weight and hepatocyte hypertrophy; findings are not considered to be adverse and a NOAEL of 1000 mg/kg bw/d is determined for this study.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Fatty acids, tall-oil, epoxidized,2-ethylhexylesters (ETP) on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

ETP was administered once daily orally (by gavage) to males for at least 28 days and to female rats throughout the prepairing and pairing periods until day 3 of lactation.

The following dose levels were applied:

Group 1:             0 mg/kg body weight/day (vehicle control)

Group 2:            100 mg/kg body weight/day

Group 3:             300 mg/kg body weight/day

Group 4:             1000 mg/kg body weight/day

A standard dose volume of 2 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil).

The following results were obtained:

Parent animals:

General Tolerability

No mortalities were observed. No test-item related effects were seen. Neither food consumption nor body weight development were affected at any dosage.

Reproduction Data:

Fertility rate was high resulting in at least 9 litters per group for evaluation of reproduction data. There were no treatment-related effects on precoital time, fertility indices, mean duration of gestation, number of implantations, post-implantation loss, pup survival or litter size from birth through to scheduled pup sacrifice on Day 4 post partum at any dosage.

Clinical Laboratory Investigations:

The assessment of clinical chemistry and haematology parameters indicated no differences between animals treated with the test item and vehicle controls.

Terminal Examinations:

During necropsy of parent animals no test item-treated findings were noted. For males treated at 300 and 1000 mg/kg/day, mean absolute and relative liver weights were dose-dependently increased.

Histopathology:

The following findings distinguished test item-treated animals from vehicle controls:

Liver- Minimal hepatocellular hypertrophy in animals treated at 1000 mg/kg/day. This change was considered to represent an adaptive reaction most likely induced by an increased biotransformation of the test item. Therefore, it was not assessed as an adverse effect.

Thyroid- Increased incidence of minimal follicular cell hypertrophy in animals treated at 1000 mg/kg/day. No test item- related histopathological findings were noted in the reproductive organs of either sex. In particular, the assessment of the integrity of the spermatogenetic cycle did not reveal any evidence of impaired spermatogenesis.

Litter Data:

No test item-related abnormal findings were noted for pups at first litter check or during the first 4 days post partum. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.

Mean pup weights on day 0 and day 1 post partum were unaffected by treatment with the test item. Mean pup weight development during the first 4 days post partum was unaffected by treatment with the test item.

F1 Pups Necropsy findings:

No test item-related macroscopic findings were noted during necropsy of F1 pups.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A modern guideline-compliant screening study with the read-across substance ETP is available. Older one-year studies in the rat and dog with the read-across substance ESBO are also available.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral data with the read-across substances ETP (ethylhexyl esters of epoxidised tall oil) and ESBO (epoxidised soybean oil) are available. A one-year dietary study in rats and dogs with ESBO at dose levels of up to 5% did not reveal any adverse effects; findings were limited to elevated liver weight at high dose levels. A screening study performed with ETP similarly identified increased liver weight and hepatocyte hypertrophy at high dose levels as the only effects of treatment.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study is of high quality, GLP and fully compliant with the OECD guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Justification for classification or non-classification

The available data show low toxicity and do not trigger classification for repeated dose toxicity according to EU criteria.