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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant study completed in accordance with current guidelines - OECD 422. Only minor deviations occurred with no notable effect on the outcome of the investigations - see 'Overall comments' for details. The study was conducted with ETP rather than ESBO but read-across between the two oils is considered appropriate.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
see 'Overall Remarks'
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
TEST ITEM
- Name of test material (as cited in study report): Fatty acids, tall-oil, epoxidized,2-ethylhexylesters
- Physical state: Liquid
- Analytical purity: 100 %
- Lot/batch No.: 502196
- Expiration date of the lot/batch: 10th May 2005
- Stability under test conditions: Stable for at least 7 days, when stored at room temperature
- Storage condition of test material: At room temperature 20 ± 5 °C

Test animals

Species:
rat
Strain:
other: HanBrl:WIST (SPF)
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
100 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
300 mg/kg body weight/day
Basis:
other: gavage
Remarks:
Doses / Concentrations:
1000 mg/kg body weight/day
Basis:
other: gavage
No. of animals per sex per dose:
40 males, 10 per group
40 females, 10 per group
Please see Table 1 for more information
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.

Examinations

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

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
One female in group 4 died during the blood sampling procedure. This was considered an accident. No further deaths occured.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female in group 4 died during the blood sampling procedure. This was considered an accident. No further deaths occured.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was slightly higher in the male group 4. This was incidental and of no toxicological relevance.
Food efficiency:
not examined
Description (incidence and severity):
Test substance was administered by oral gavage
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
Test substance was administered by oral gavage
Ophthalmological findings:
not examined
Description (incidence and severity):
Not required in range-finding examination
Haematological findings:
no effects observed
Description (incidence and severity):
No effects
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No effects
Urinalysis findings:
not examined
Description (incidence and severity):
Not required
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Locomotor activity was statistically higher in group 3 and 4 males than in the respective control. This was attributable to normal biological variation.
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
Description (incidence and severity):
No effects
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please see below
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Not required
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. See table 3 for further information.

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.

Please see table 2 for F0 animal breeding statistics.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
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 (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Higher doses resulted in increased liver weight

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

 

Applicant's summary and conclusion