Registration Dossier

Diss Factsheets

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: Guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report date:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: 51 FR 40329, Section 795.260
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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
Constituent 2
Reference substance name:
2-ethylheaxanoic acid
IUPAC Name:
2-ethylheaxanoic acid
Details on test material:
- Name of test material (as cited in study report): 2-ethylhexanoic acid
- Analytical purity: 99.9 +/- 0.05%
- Stability under test conditions: stable over a seven day period in open feeders; 35 days under refrigeration
- Storage condition of test material: refrigerated

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA, USA
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: males: 140 +/-4 g; females: 100 +/- 3 g
- Housing: groups of 5 segregated by sex
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23 deg C (reported as 70-74 deg F)
- Humidity (%): 45-58%
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: diets were prepared containing 0.0%, 0.1%, 0.5%, or 1.5% of the test material.

DIET PREPARATION
- Rate of preparation of diet (frequency): multiple batches prepared; no individula batch was used for a period exceeding the 35 day stability.
- Mixing appropriate amounts with (Type of food): AGWAY Prolab animal diet (RMH 3200)
- Storage temperature of food: kept refrigerated
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
GC/FID
Duration of treatment / exposure:
non-recovery animals: 91-93 days
recovery animals: 93 days
Frequency of treatment:
7 days in diet
Doses / concentrationsopen allclose all
Dose / conc.:
71 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
360 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
1 068 mg/kg bw/day (actual dose received)
Remarks:
females
Dose / conc.:
61 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
303 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
917 mg/kg bw/day (actual dose received)
Remarks:
males
Dose / conc.:
1.5 other: % nominal in diet
Remarks:
non-recovery group and 28 d recovery group
Dose / conc.:
0.5 other: % nominal in diet
Remarks:
non-recovery group
Dose / conc.:
0.1 other: % nominal in diet
Remarks:
non-recovery group
Dose / conc.:
0 other: % nominal in diet
Remarks:
non-recovery group and 28 d recovery group
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
males and females
No. of animals per sex per dose:
Groups of 10 male and 10 female Fischer 344 rats were fed diets containing either 0.0, 0.1, 0.5 or 1.5% 2-ethylhexanoic acid (EHA) for 13 wk. Additional groups of 10 male and 10 female rats were fed either 0.0 or 1.5% EHA for 13 wk followed by a 4-wk recovery (non-treatment) period.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: doses were selected following range-finding studies
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery, non-treatment
- Post-exposure recovery period in satellite groups: 28-days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every workday afternoon and on mornings that body weight measurements were not conducted.
- Cage side observations: observation included, but was not limited to, examination of the hair, skin, eyes, motor activity, feces, and urine

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: mornings of body weight measurement

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 4, 7 and at least once each week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed weight collected Day 4 and 7 and at least once weekly thereafter
- Food consumption for each animal determined and 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: No data

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of the study and during the last week of exposure
- Dose groups that were examined: all animals prior to the start of the study; five male and five female non-recovery animals from each dose level were examined during the last week of exposure.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy of non-recovery groups
- Anaesthetic used for blood collection: Yes (identity) CO2
- Animals fasted: Yes- overnight
- How many animals: 5 animals/sex/dose level, including controls
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy of non-recovery groups
- Animals fasted: Yes- overnight
- How many animals: 5 animals/sex/dose level, including controls
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: week prior to study termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: electron microscopy: Sections of liver were taken from five animals from each dose group, including the recovery groups.
Sacrifice and pathology:
Rats were fasted overnight, anesthetized with CO2, and exsanguinated by severing the posterior vena cava after collecting blood for analysis.
The liver, kidneys, adrenal glands, testes, ovaries, and brain were weighed.
Paired organs were weighed together. Organ/body weight and organ/brain weight ratios were calculated.
The following organs were fixed in 10% buffered formalin: trachea, lungs, heart, aorta, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal glands, thyroid glands, parathyroid glands, thymus, spleen, mesenteric lymph nodes, bone marrow (femoral), brain (including sections of medulla/pons, cerebellar cortex, and cerebral cortex), [cervical, mid-thoracic, and lumbar spinal cord], sciatic nerve, quadriceps femoris, testes, [epididymides], [male accessory sex glands], [male mammary gland], ovaries, vagina, uterus, Fallopian tubes, [female mammary gland], sternum with bone marrow, [femur (including articular surface)], [skin], [exorbital lachrymal glands], and gross lesions.

For non-recovery animals, all tissues not enclosed in brackets above from high-dose and control groups were examined histologically. The liver,
kidneys, lungs, target organs, and gross lesions for animals from all dose levels were examined. Histopathology was not performed on bracketed organs, because no signs of toxicity or target organ involvement was observed.

For recovery animals, histopathology was performed on the liver, kidneys, lungs, and gross lesions.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Mean values were calculated for body weight, feed consumption, and organ weights. Numerical data were evaluated for statistical significance using the following computer-generated statistical tests:
Bartlett's test (p was not analyzed statistically because animals were group housed and ate from a common container.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No significant clinical signs of toxicity occurred during the rat study.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the rat study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In rats, 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 rat 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):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption by rats 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
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. These differences were minimal, however, and did not indicate clinically significant changes in red blood cell indices.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Cholesterol levels were higher than the control values (P<=0.05) for all male rat test groups. After 28 days of recovery, cholesterol levels for male rats (1.5 % group) were comparable to control values. The only other change in serum chemistry was an increase in albumin in male rats fed 1.5% EHA; this value returned to control levels after 28 days of recovery.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No clinically significant treatment-related urinalysis changes were observed.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean relative (to body weight) liver weights for all groups of animals receiving 0.5% or 1.5% EHA were greater (P<=0.05) than the weights of the respective control groups. The mean absolute liver weights for all groups fed 1.5% EHA, and for female rats fed 0.5% EHA were also significantly greater (P<=0.05) than for the respective control groups. No other changes in liver weights were noted and following 28 days of recovery, all absolute liver weights were comparable to control values. Absolute kidney weights for rats were unaffected by EHA treatment. In female rats, however, 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% groups rather than indicative of any particular effect on the kidney. Minor, yet statistically significant (P<=0.05) decreases in absolute brain weights (1.5% female rats) were not considered biologically significant. In addition, while slight increases in relative adrenal gland, brain, and testes weights occurred for some of the groups, the differences were related to reduced terminal body weight and, therefore, were not considered to be indicative of organ specific toxicity.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related gross pathology was observed for either main-study or recovery animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hepatocyte hypertrophy was observed after 13 wk of treatment in 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 vacuolization 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.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: no mortality was observed during the study. Clinical abnormalities were minimal during the main 90-day study and the recovery period. Porphyrin tears, porphyrin nasal discharge, facial wounds, urine stained hair, and alopecia were observed without any relationship to treatment group. These abnormalities were transient and had no impact on the study.

BODY WEIGHT AND WEIGHT GAIN: No treatment-related differences for body weight or feed consumption were observed between controls and 0.5 and 0.1% males and females.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Feed consumption was reduced for both the recovery and non-recovery 1.5% animals of both sexes during the first 4 days of the study and generally remained lower than that of the controls throughout the treatment period. This reduction resulted in a slightly decreased rate of body weight gain for all animals receiving the 1.5% 2-EHA diets. At the end of the 90-day treatment period, the body weights of the 1.5% males and females were 8 and 10% (non-recovery) and 9 and 8% (recovery) lower than controls, respectively. During the recovery period, the mean consumption of basal diet for the recovery animals increased to levels equal to or slightly higher than that of controls. This increase resulted in an increase in body weight gain. By the end of the recovery phase, the body weights were only 5 and 3% lower than controls for males and females, respectively, and the difference for the females was no longer statistically significant.

FOOD EFFICIENCY: not examined

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):not examined

OPHTHALMOSCOPIC EXAMINATION: No ophthalmologic abnormalities were observed in any recovery or non-recovery animal in the pre-study examination or in the five males and five females from each non-recovery dose group when they were examined during the last week of test article exposure.

HAEMATOLOGY: Minor hematologic differences involving red blood cells were observed at the 0.5 and 1.5% dose levels for both males and females. These included lower mean corpuscular volume in the 1.5% non-recovery and recovery females; lower mean corpuscular hemoglobin in the 0.5 and 1.5% non-recovery males, 1.5% recovery males, and 1.5% non-recovery females; and lower hemoglobin concentration in the 0.5% non-recovery females. The significance of these differences is obscure since all of the differences are minimal, the differences do not indicate any clinically significant change in the blood picture of the animals, and none of the changes were accompanied by evidence of anemia.

CLINICAL CHEMISTRY: The principal effects of the test article involved the liver. Exposure to 2-EHA resulted in a dose-dependent increase of cholesterol for males at all dose levels. Cholesterol levels for the males on the 0.1, 0.5, and 1.5% diets were increased 25, 42, and 78% relative to the controls. A similar change was not observed for cholesterol levels in females. Following the recovery period, cholesterol levels returned to normal. Triglycerides were not similarly altered. The alteration in cholesterol levels in males only may indicate a normal sex-dependent difference in fatty acid metabolism, since 2-EHA in the diet may act as a nutrient. Increased albumin concentrations in the serum may also indicate an effect on the liver; this difference was seen only at 1.5% 2-EHA in males and was not seen after the recovery period.

URINALYSIS: Urine volume was reduced for all females given 2-EHA for 90 'days and urine specific gravity was slightly increased for the 1.5% females. Similar effects were not observed for the males or for the females examined after the recovery period. Most significantly, urine osmolality did not differ between animals given 2-EHA and controls, indicating that 2-EHA did not interfere with the ability of the kidney to concentrate urine. Serum urea nitrogen levels were marginally higher than controls only for the 1.5% males at 90 days; at the end of the recovery period a similar difference was not present. The difference in urea nitrogen was not a clinically significant elevation.

ORGAN WEIGHTS: The absolute liver weights of the 0.5 and 1.5% animals after 90-days of 2-EHA exposure were 6 and 32% (males) and 7 and 19% (females) higher than those of the controls. This increase occurred in spite of the reduced body weight of the 1.5% animals. Liver weights relative to body and brain weights were also greater at the two higher doses. These effects were all statistically significant except for the absolute liver weights and liver/brain weights for the 0.5% male group. After the recovery period, the effect on liver weight was largely reversed except that the liver/body weight difference persisted for the 1.5% males. Weight differences observed in other organs were also likely to be related to growth retardation rather than toxicity. The weight differences observed in the brain are consistent with those seen in animals in which growth has been retarded. This difference was no longer evident in the 1.5% females by the end of the recovery period, but was still evident in the 1.5% males. The higher mean relative testes/body weights observed for the 0.5 and 1.5% males were consistent with effects observed in animals in which body growth has been retarded but testicular growth has been spared; the difference was still present in the 1.5% recovery males.

GROSS PATHOLOGY: No treatment-related changes were observed in non-recovery or recovery animals at the time of necropsy at any dose level for either sex.

HISTOPATHOLOGY: NON-NEOPLASTIC: At necropsy, the livers from treated animals were indistinguishable from the controls at 90 days and after the recovery period. Histologic examination of the livers revealed hypertrophy of the hepatocytes and a reduction of the number of small cytoplasmic vacuoles in hepatocytes from males and females fed 1.5% 2-EHA for 90 days and males fed 0.5% for the same length of time. The severity of both lesions was dose-dependent. Hepatocyte hypertrophy and decreased hepatocyte vacuolization were not seen following the recovery period indicating that these were reversible effects.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Minimal hyperplasia of bile ducts was found at both the 0.0 and 1.5% dose level in both sexes of recovery animals. The incidence for males was 4 of 10 at 1.5% versus 1 of 10 in the controls, and for females was 1 of 10 at 1.5% versus 3 of 10 at 0.0%. Bile duct hyperplasia was considered minimal when no more than three groups of newly formed ducts were observed in any of the liver sections. Because hyperplasia of this type and severity has been observed in control rats in other studies and because the incidence of hyperplasia can vary considerably, the differences in frequency of bile duct hyperplasia observed in this study were not considered biologically significant.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
303 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: growth retardation
Dose descriptor:
NOAEL
Effect level:
360 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: growth retardation
Dose descriptor:
NOEL
Effect level:
71 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: liver enlargement; considered primarily an adaptive change rather than a toxic effect.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The no-observed-adverse-effect-level (NOAEL) was considered to be 0.5% 2-EHA in the diet or 303 mg/kg/day for the males and 360 mg/kg/day for the females. This NOAEL was based on growth retardation at the highest dose level. The no-observed-effect-level (NOEL) for liver enlargement was considered to be 0.1% 2-EHA in the diet or 71 mg/kg/day for the females; liver enlargement was considered primarily an adaptive change rather than a toxic effect. There was no NOEL for males defined based on the increased cholesterol levels observed at all treament levels. Recovery following exposure to 2-EHA in the diet was essentially complete after 28 days. The 5% difference in mean body weight between the control and the 1.5% males at the end of the recovery period was the only effect which had not completely reversed.
Executive summary:

Groups of 10 male and 10 female rats were fed diets containing 2-ethylhexanoic acid (2-EHA) at concentrations of 0.0, 0.1, 0.5, or 1.5% for 90 days. Additional groups of 10 male and 10 female rats were fed either 0.0 or 1.5% 2-EHA for 90 days followed by a 28-day recovery (non-treatment) period. The control diet and the three 2-EHA diets provided dose levels of 0, 61, 303, or 917 mg/kg/day for males and 0, 71, 360, or 1068 mg/kg/day for females. Consumption of 2-EHA diets did not result in mortality, significant clinical abnormalities, or ophthalmologic abnormalities.

The 1.5% diet resulted in reduced feed consumption for male and female rats. During the recovery period, feed consumption in the group previously administered the 1.5% diet increased to a level comparable with or higher than controls. The lower two concentrations of diet did not alter feed consumption. The 1.5% diet was associated with reduced body weight gain. Mean body weight was 8% (males) and 10% (females) less than the control body weight for the non-recovery animals at the end of the treatment period. Similarly, mean body weight was 9% (males) and 8% (females) less for the recovery animals at the end of the treatment period. At the end of the 28-day recovery period, the body weights for the 1.5% males and females, respectively, were 5% and 3% lower than controls. The mean body weights for the 0.5 and 0.1% males and females were comparable to controls.

Slight hematologic differences involving red blood cells were observed at the 0.5 and 1.5% dose levels for both males and females, but these changes did not indicate any clinically significant change.

The principal effects of the test article involved the liver or metabolic processes associated with the liver. The 0.5 and 1.5% diets were associated with increased liver weight and histologic changes including hypertrophied hepatocytes and hepatocytes with reduced cytoplasmic vacuolization. Cholesterol levels were increased at all dose levels in males, but not in females. Liver effects were reversible upon removal of 2-EHA from the diet. At the end of the recovery period, only the liver weight relative to body weight for the males was statistically significant; this difference was due to lower body weights for the males. Cholesterol returned to normal levels during the recovery period.

Urinalysis results were unremarkable except for decreased urine volume for all groups of treated females and increased urine specific gravity for the 1.5% females after the 90-day treatment period. Urine osmolality was comparable between treated and control groups, indicating that the kidneys were able to concentrate urine normally. At the end of the recovery period, no differences were observed between treated and control animals. Male rats did not have similar differences at either collection time.

Kidney, testes, and brain weight differences were observed, but the differences were most likely a reflection of lower mean body weight, rather than target organ effects. Adrenal and ovarian weights were not altered by 2-EHA exposure.

The no-observed-adverse-effect-level (NOAEL) was considered to be 0.5% 2-EHA in the diet or 303 mg/kg/day for the males and 360 mg/kg/day for the females. This NOAEL was based on growth retardation at the highest dose level. The no-observed-effect-level (NOEL) for liver enlargement was considered to be 0.1% 2-EHA in the diet or 71 mg/kg/day for the females; liver enlargement was considered primarily an adaptive change rather than a toxic effect. There was no NOEL for males defined based on the increased cholesterol levels observed at all treament levels. Recovery following exposure to 2-EHA in the diet was essentially complete after 28 days. The 5% difference in mean body weight between the control and the 1.5% males at the end of the recovery period was the only effect which had not completely reversed.