Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
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:
reference to same study

Data source

Referenceopen allclose all

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 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 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:
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

Reference
Name:
Unnamed
Type:
Constituent
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:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals 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.
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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
180, 885, and 2728 mg/kg bw/day of 2 ethylhexanoic acid (male mice)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
205, 1038, and 3139 mg/kg bw/day of 2 ethylhexanoic acid (female mice)
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)
180 mg/kg bw/day: 10 males
205 mg/kg bw/day: 10 females
885 mg/kg bw/day: 10 males
1038 mg/kg bw/day: 10 females
2728 mg/kg bw/day: 10 males (recovery group: 10 males)
3139 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:
- 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: 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, 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) of 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 euthanized. 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 less for animals consuming diets containing 1.5% EHA compared with controls.
- Mean body weights were 5% (P ≤ 0.05) and 14% (P ≤ 0.05) lower for male and female mice, respectively, in the 1.5% EHA groups at the end of the treatment period compared with the control group.
- Mean body weight of female mice receiving 1.5% EHA was statistically (P ≤ 0.05) lower than the control group mean beginning after the first week on test through the recovery period.
- Mean body weight of the 0.5% females was statistically lower (P ≤ 0.05) than the control group mean at study termination.
- Body weight gains were unaffected in mice consuming diets containing 0.1% EHA.
- Body weight decrements for the male and female high-dose groups persisted throughout the recovery phase and were statistically significant (P ≤ 0.05) at all time intervals with the exception of the last interval day (119) for males.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
- EHA diets provided dose levels of 180, 885 or 2728 mg/kg/day for male mice and 205, 1038 or 3139 mg/kg/day for female mice.
- Mean feed consumption by mice on the 1.5% diet was equal to or slightly less than that of the control group throughout the study. Feed consumption by other dose groups was unaltered from control values.

OPHTHALMOSCOPIC EXAMINATION
- Lack of treatment -related changes.

HAEMATOLOGY
- No haematological differences were seen for mice.

CLINICAL CHEMISTRY
- Cholesterol levels were higher than the control values (P ≤ 0.05) for all male and female mice fed either 0.5 or 1.5% EHA. After 28 days of recovery, cholesterol levels for both sexes of mice (1.5% group) were comparable to control values.
- Triglyceride concentrations were lower (P ≤ 0.05) for the 1.5% male mice and for the 0.5% and 1.5% female mice, compared with control values. After recovery, no differences were seen in female mice, but males still had a 27% lower (P ≤ 0.05) mean triglyceride level than the control group. The difference in triglyceride levels for the male recovery group appears to be an artefact resulting from the higher control triglyceride value.
- Bilirubin was lower (P ≤ 0.05) for both 1.5% male and female mice and 0.5% female mice after 13 weeks of treatment, bilirubin levels returned to normal after recovery for female mice, but continued to be lower for male mice.
- Alanine aminotransferase for the 1.5% male mice was higher (P ≤ 0.05) at the 13-week sacrifice. The mean alanine aminotransferase level was lower (P ≤ 0.05) after recovery, but this was not considered to be biologically significant.

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 all groups fed 1.5% EHA and for male mice 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 mice 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 mice) and absolute adrenal weights (0.5% and 1.5% female mice 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 mice, while proximal renal tubule cytoplasmic basophilia (males and females) and hyperkeratosis of the mucosa of the forestomach (males only) were observed in mice fed 1.5% EHA. The hepatocyte hypertrophy occurred in males and females fed 1.5% EHA, and in male mice 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. Moderate eosinophilia of the hepatocyte cytoplasm was an additional effect seen only in the high-dose mice groups. The liver changes were reversible since only two of 20 mice showed hepatocyte hypertrophy after 28 days of recovery, and the severity was reduced compared with that observed at the end of exposure.
- Minimal to minor cytoplasmic basophilia of the proximal convoluted tubules in the kidneys of high-dose mice was observed after 13 weeks. These same cells contained small numbers of cytoplasmic vacuoles, and nuclei were slightly enlarged and vesicular with marginated chromatin. Kidney effects were seen in four of 10 male and four of 10 female mice from the 1.5% group. Kidney changes were not seen at lower dose levels. Renal changes were absent for recovery animals.
- Acanthosis and hyperkeratosis of the non-glandular forestomach were noted only in the 1.5% male group of mice necropsied after 13 weeks.
- No testicular changes were noted.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Based on body weight retardation and liver changes.
Dose descriptor:
NOAEL
Effect level:
205 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Based on body weight retardation and liver changes.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL (male mice): 180 mg/kg/day (actual received)(based on body weight retardation and liver changes.)
NOAEL (female mice): 205 mg/kg/day (actual received)(based on body weight retardation and liver changes.)


No mortality or significant clinical signs of toxicity were observed during the study. Body weights and food consumption of mice 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. After 13 week, lower triglyceride levels occurred in male mice fed 1.5% EHA and female mice fed 0.5 or 1.5% EHA, but not in other groups. Cholesterol levels were higher in male and female mice 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 mice 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 mice following subchronic dietary exposure at concentrations up to 1.5% in feed.