Barium bis(2-ethylhexanoate)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Materials, methods and results well described/presented in text and tables

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

2-Ethylhexanoic acid: Subchronic oral toxicity studies in the rat and mouse

Author:

Juberg, D. R. et al.

Year:

1998

Bibliographic source:

Food and Chemical toxicology, 36: 429 - 436.

Reference 2

Reference Type:

publication

Title:

Subchronic oral toxicity studies of 2-ethylhexanoic acid in the rat and the mouse

Author:

Topping, D. C. et al.

Year:

1989

Bibliographic source:

The Toxicologist 9, 109.

Reference 3

Reference Type:

publication

Title:

TOXICOLOGICAL EVALUATION No. 275 2-ethylhexanoic acid 06/00

Author:

BG Chemie

Year:

2000

Bibliographic source:

BG Chemie InfoCenter TOXIKOLOGISCHE BEWERTUNGEN (www.bgchemie.de)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: TSCA (1992) health Effects Testing Guidelines for Subchronic Oral Toxicity Studies. Title 40, CFR 798. 2650.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexanoic acid

EC Number:

205-743-6

EC Name:

2-ethylhexanoic acid

Cas Number:

149-57-5

Molecular formula:

C8H16O2

IUPAC Name:

2-ethylhexanoic acid

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): 2-Ethylhexanoic acid (EHA; obtained from Eastman Chemical Company (Kingsport, TN, U.S.A.))
- Analytical purity: 99.9 ± 0.05%
The mass spectrum was consistent with the structure of EHA.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - CDF®F344/CrlBR)
- Source: Charles River Laboratories (Wilmington, MA, USA)
- Age at study initiation: approximately 6 weeks
- Housing: animals were housed in a separate room in an American Association for Accreditation of Laboratory Animal Care-accredited facility. Husbandry and environmental conditions were in compliance with the NIH Guide to the Use and Care of Animals (86-23)
- Diet (ad libitum): certified feed (Agway® Prolab TM Animal Diet RMH 3200)
- Water (ad libitum): water
- Acclimation period: approximately 2 weeks, determined to be healthy prior to testing

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test substance was mixed with ground certified chow using a Hobart cafeteria-type food mixer (Model M-802) to yield target EHA concentrations. The test substance was added directly (no vehicle) to chow in the mixing bowl and this mixture was then blended for at least 30 minutes. The control chow was treated in an identical manner (without the addition of EHA). Diets were prepared monthly and the concentrations of test diets were verified analytically.
The actual dose received was calculated taking into account the amount of ingested food, concentration of the a.i. in food and the bodyweight of the test animals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

13 consecutive weeks

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
61, 303, and 917 mg/kg bw/ day of 2 ethylhexanoic acid (male rats)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
71, 360, and 1068 mg/kg bw/day of 2 ethylhexanoic acid (female rats)
Basis:
actual ingested

No. of animals per sex per dose:

0 mg/kg bw/day: 10 males / 10 females (recovery group: 10 males / 10 females)
61 mg/kg bw/day: 10 males
71 mg/kg bw/day: 10 females
303 mg/kg bw/day: 10 males
360 mg/kg bw/day: 10 females
917 mg/kg bw/day: 10 males (recovery group: 10 males)
1068 mg/kg bw/day: 10 females (recovery group: 10 females)

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: 10 animals per sex per group from the high-dose and control groups, included to assess recovery, were continued on the control diet for an additional 28 - 29 days (recovery period).

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice each workday for clinical signs of toxicity and daily for mortality
- Cage side observations checked: clinical signs of toxicity included examination of the hair, skin, eyes, motor activity, faeces and urine.
Clinical observations for recovery animals were identical to those performed on animals during the test phase of the study.

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: twice during the first week and at least once weekly thereafter
Clinical observations for recovery animals were identical to those performed on animals during the test phase of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, usually measured twice weekly throughout the study
- Mean daily diet consumption calculated as g food/kg body weight/day: No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Clinical observations for recovery animals were identical to those performed on animals during the test phase of the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All rats were examined for ocular effects prior to the start of the study. During the last week of test article exposure, the eyes of five male and five female animals from each dose level were examined.
Clinical observations for recovery animals were identical to those performed on animals during the test phase of the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: Yes, CO2
- Animals fasted: Yes, all animals were fasted overnight.
- How many animals: five animals per sex from each dose level
- Parameters examined: haemoglobin concentration, haematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time, and examination of the blood smears for cellular morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes, all animals were fasted overnight.
- How many animals: five animals per sex from each dose level
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, total bilirubin, total protein, albumin, creatinine, urea nitrogen, glucose, γ-glutamyl transpepitdase, triglycerides, cholesterol, sodium, potassium, chloride, calcium and phosphorous

URINALYSIS: Yes
- Time schedule for collection of urine: urine samples from five animals per sex per group (non-recovery groups) were analysed the week prior to termination of the treatment part (13-week) fo the study and at termination of the recovery period.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: specific gravity, osmolality, volume of urine, glucose, ketones, pH, protein and haemoglobin

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
At then end of 13 weeks, 10 animals per group per sex were euthanised. All animals were anaesthetised with CO2, and exsanguinated form the posterior vena cava after collecting blood for analysis.Complete necropsies were performed on all animals.
The liver, kidneys, adrenal glands, testes, ovaries and brain were weighed. Paired organs were weighed together. All tissues listed in the TSCA Health Effects Testing Guidelines for Subchronic Oral Toxicity Studies (TSCA, 1992)* were fixed in 10% buffered formalin and evaluated for the high dose and control animals. The liver, kidneys, lungs and gross lesions from animals of all dose levels were examined. For the recovery groups, histopathology was performed on livers, kidneys, lungs and gross lesions. All tissues were embedded in paraffin, sectioned at 5 - 6 microns, and stained with haematoxylin-eosin.

*Reference:
-TSCA (1992) health Effects testing Guidelines for Subchronic Oral Toxicity Studies. Title 40, CFR 798. 2650.

Other examinations:

no data

Statistics:

Numerical data were evaluated for statistical significance using the following computer-generated statistical tests: Bartlett's test (P ≤ 0.01), one-way analysis of variance (ANOVA) (P ≤ 0.05), and Duncan's multiple range test (P ≤ 0.05).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality or clinical signs of toxicity occurred during the study.

BODY WEIGHT AND WEIGHT GAIN
- Body weight gain was slightly lower for animals consuming diets containing 1.5% EHA compared with the control groups.
- Mean body weights for the 1.5% male and female groups were respectively 8% and 10% lower (P ≤ 0.05) at the end of the treatment period and were statistically (P ≤ 0.05) lower than the control group beginning after the first week on test.
- Mean body weights of rats consuming diets containing 0.5% or 0.1% EHA were unaffected compared with controls.
- During the recovery phase, body weight gain for animals that had received 1.5% EHA increased so that by the end of the recovery period, the mean body weight for males was only 5% less (P ≤ 0.05), and for females only 3% less (P ≤ 0.05), than their respective control groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- When calculated as a time-weighted average, the three EHA diets provided dose levels of 61, 303 or 917 mg/kg/day for male rats and 71, 360 or 1068 mg/kg/day for female rats.
- Feed consumption was initially reduced between 19 and 26% in animals consuming the 1.5% diet relative to that of the control group. From day 4 to the end of the treatment period for that group, the average feed consumption was 3 - 5% lower in males, and 8 - 10% lower in females when compared with their respective control groups.
- No differences in feed consumption were seen for the 0.1 and 0.5% groups or for the 1.5% recovery group compared with controls.

OPHTHALMOSCOPIC EXAMINATION
- Lack of treatment -related changes.

HAEMATOLOGY
- Minor red blood cell differences (reductions in mean corpuscular haemoglobin and mean corpuscular volume ) were observed at the 0.5 and 1.5% dose levels for male and female rats (no indication of clinically significant changes in red blood cell indices)

CLINICAL CHEMISTRY
- Cholesterol levels were higher than the control values (P ≤ 0.05) for all male rat test groups and for female rats fed either 0.5 or 1.5% EHA. After 28 days of recovery, cholesterol for male rats (1.5% group) were comparable to control values.
- Triglyceride concentrations for rats were not significantly different from control values.
- An increase in albumin in male rats fed 1.5% EHA; this value returned to control values after 28 days of recovery.

URINALYSIS
- No clinically significant treatment-related urinalysis changes were observed.

ORGAN WEIGHTS
1) Liver
- Mean relative (to body weight) liver weights for groups receiving 0.5% or 1.5% EHA were greater (P ≤ 0.05) than the weights of the respective control groups.
- Mean absolute liver weights for groups fed 1.5% EHA and for female rats fed 0.5% EHA were also significantly greater (P ≤ 0.05) than for te respective control groups.
- Following 28 days of recovery, all absolute liver weights were comparable to control values.
2) Kidney
- Absolute kidney weights were unaffected by EHA treatment.
- In female rats greater relative kidney weights were noted for both the 1.5 and 0.5% diet groups. Greater relative kidney weight differences were reflective of lower body weights for the 1.5% group rather than indicative of any particular effect on the kidney.
3) Brain, adrenal gland and testes
- Minor, statistically significant (P ≤ 0.05) decreases in absolute brain weights (1.5% female rats) were not considered biologically significant.
- Slight increases in relative adrenal gland, brain, and testes weights occurred for some of the groups (differences were related to reduced terminal body weight and, therefore, were not considered to be indicative of organ specific toxicity).

GROSS PATHOLOGY
- No treatment-related gross pathology was observed for either main-study or recovery animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Hepatocyte hypertrophy was observed after 13 weeks of treatment for rats. The hepatocyte hypertrophy occurred in males and females fed 1.5% EHA, and in male rats fed 0.5% EHA, but with decreased incidence and severity. The number of EHA-treated animals with hepatocyte cytoplasmic vacuolisation decreased as both the EHA dose and incidence of hypertrophy increased. No hepatic lesions were observed in rats after recovery.
- Renal changes were not observed for rats.
- No testicular changes were noted.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on liver changes, which in the authors assessment were not signs of toxicity but rather adaptive change.

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on liver changes, which in the authors assessment were not signs of toxicity but rather adaptive change.

3

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on reduced feed consumption and decreased rate of body weight gain, reversible within 14-day recovery.

4

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on reduced feed consumption and decreased rate of body weight gain, reversible within 14-day recovery

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOAEL (male rats): 61 mg/kg/day (actual received)(based on liver changes, which in the authors assessment were not signs of toxicity but rather adaptive change.)
NOAEL (female rats): 71 mg/kg/day (actual received)(based on liver changes, which in the authors assessment were not signs of toxicity but rather adaptive change.)
LOAEL (male rats): 303 mg/kg/day (actual received)(based on reduced feed consumption and decreased rate of body weight gain, reversible within 14-day recovery)
LOAEL (female rats): 360 mg/kg/day (actual received)(based on reduced feed consumption and decreased rate of body weight gain, reversible within 14-day recovery)

No mortality or significant clinical signs of toxicity were observed during the study. Body weights and food consumption of rats fed 1.5% EHA were lower beginning after the first week of treatment, consistent with a reduction in food consumption. Other groups were unaffected by treatment. Cholesterol levels were higher in all male rat test groups and in female rats fed either 0.5 or 1.5% EHA, although this effect was reversible following a 28-day recovery period. The principal effects of EHA involved the liver or metabolic processes associated with the liver. The 0.5 and 1.5% diets in rats were associated with increased relative liver weight and histological changes in hepatocytes, specifically hepatocyte hypertrophy and reduced cytoplasmic vacuolization. Observed histopathological and clinical pathological changes were reversible following recovery. These results indicate that EHA does not produce persistent, overt toxicity in rats or mice following subchronic dietary exposure at concentrations up to 1.5% in feed.