Registration Dossier

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
Study period:
1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near guideline study, with minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OTS 795.2600 (Subchronic Oral Toxicity Test)
GLP compliance:
yes
Remarks:
Health and Environmental Laboratories, Eastman Kodak Company
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.9% (±0.05%; GC)
- The test material is stable in open feeders for at least 7 days (GC)

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 140 (± 4) g; females: 100 (± 3) g
- Housing: 5 animals/cage
- Diet (e.g. ad libitum): Agway Prolab Animal Diet (RMH 3200), certified ground chow; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-74
- Humidity (%): 45-58
- Photoperiod (hrs dark / hrs light): 12/12

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. The diets were frozen in closed amber glass bottles until used. The test material was stable for at least 35 days in the diets when refrigerated. Stability, homogeneity and concentration analyses were conducted using GC.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed concentrations (mean ± SD; GC): 0.091 (± 0.004), 0.45 (± 0.02) and 1.45 (± 0.05)%
Duration of treatment / exposure:
91-93 days
Frequency of treatment:
7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 0.1, 0.5 and 1.5%. 28-day recovery groups: 0 and 1,5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 0.091, 0.45 and 1.45% (recovery groups: 0 and 1.45%)
Basis:
other: analyzed in diet
Remarks:
Doses / Concentrations:
0, 61, 303 and 917 mg/kg/day for males (recovery: 0 and 908 mg/kg/day); 0, 71, 360 and 1068 mg/kg/day for females (recovery: 0 and 1044 mg/kg/day)
Basis:
other: actual dose
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based on a 14-day feeding study in rats
- Post-exposure recovery period in satellite groups: 27-28 days
Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Detailed observations: on the mornings of body weight measurement.
- Cage side observations: every workday afternoon and on mornings on which body weights were not collected. Animals were checked for mortality on weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 4, 7, and at least once weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed weights (g) were collected on days 4, 7, and at least once weekly thereafter.

OPHTHALMOSCOPIC EXAMINATION: Yes, using an indirect ophthalmoscope after dilation of the pupils with 1% Mydriacyl.
All rats were examinde prior to the start of the study. During the last week of exposure, five male and five female non-recovery animals from each dose level were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (CO2 anaesthesia)
- Animals fasted: overnight
- How many animals: five animals per sex, per dose.
- Parameters: Non-recovery groups: hemoglobin concentration, hematocrit, 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. Recovery groups: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, platelet count, red blood cell indices.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: overnight
- How many animals: five animals per sex per dose
- Parameters: Non-recovery groups: aspartate aminotransferase, alanine aminotransferase, total bilirubin, total protein, albumin, creatinine, urea nitrogen, glucose, gamma glutamyl transpeptidase, triglycerides, cholesterol, sodium, potassium, chloride, calcium and phosphorus. Recovery groups: albumin, urea nitrogen, triglycerides, cholesterol.

URINALYSIS: Yes
In the week prior to termination of exposure, five males and five females from each dose group were place in metabolism cages for 24-hour urine collections. Parameters: Non recovery-groups: specific gravity, osmolality, volume, glucose, bilirubin, ketones, blood, protein, urobilinogen, nitrite, leukocytes and pH. Recovery-groups: specific gravity, osmolality and volume.

Sacrifice and pathology:
Organ weights: liver, kidneys, adrenal glands, testes, ovaries, brain.
Histopathology: all non-recovery high-dose and control animals: 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), sciatic nerve, quadriceps femoris, testes, ovaries, vagina, uterus, fallopian tubes, sternum with bone marrow, and gross lesions.
The liver, kidneys, lungs, target organs, and gross lesions for animals from all dose levels were examined.
Because no signs of toxicity or target organ involvement was observed, no histopathology was performed on cervical/mid-thoracic/lumbar spinal cord, epididymides, male accessory sex glands, male mammary gland, female mammary gland, femur (including articular surface), skin, and exorbital lachrymal glands.
For recovery animals, histopathology was performed on the liver, kidneys, lungs, and gross lesions.
Other examinations:
Sections of the liver were prepared for electron microscopy and stored for possible future examination.
Statistics:
Means were calculated for body weight, feed consumption, and organ weights. Numerical data were evaluated using Bartlett's test, one-way ANOVA, and Duncan's multiple range test. Feed consumption was not analyzed statistically because animals were group housed.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at 1.5% substance in diet
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
reduced at 1.5% substance in diet
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
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
increased cholesterol level in males (reversible)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
liver weight increased (reversible)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hypertrophied hepatocytes
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
- Urine-soaked inguinal, abdonimal, scrotal, and/or thigh haircoats, discolored yellow, and/or unkempt: observed periodically during treatment in males and frequently in females, at all dose levels.
No mortality occured during the study.

BODY WEIGHT AND WEIGHT GAIN
A reduced mean body weight was observed in males and females of the high-dose group (8% and 10% below control weights at necropsy, respectively). Because of a caging problem, decreased body weights were observed in the mid-dose females on day 21. No effects on body weights were observed in the other groups.
During the recovery phase, the rate of body weight gain for the high-dose animals increased, but was still slightly below control level at study termination.

FOOD CONSUMPTION
During the first few days of the study, average feed consumption was moderately reduced in the high-dose group. Average consumption from day 4 to the end of the treatment period for the 1.5% males and females was 3.5 and 8.5% (non-recovery) and 4.7 and 9.5% (recovery) lower than controls, respectively. During the recovery period, average food intake was 3.7% and 0.8% higher than controles for the males and females of the exposed groups, respectively. Feed consumption was comparable to control levels in the low- and mid-dose groups.

HAEMATOLOGY
Mean corpuscular hemoglobin (MCH) was very slightly decreased in males of the mid- and high-dose groups. Females of the high-dose group had lower values for MCH and mean corpuscular volume (MCV) and the mid-dose females had a decreased mean hemoglobin concentration. Slight poikilocytosis was observed at all dose levels for males and at the mid- and high-dose for the females. Additional erythrocyt changes consisted of spherocytosis (two high-dose males), microcytosis (one high-dose female) and decreased polychromasia (in high-dose males and control, low- and high-dose females).
In the high-dose recovery group, males showed a lower MCH and females a decreased MCV, relative to controls.

CLINICAL CHEMISTRY
Cholesterol levels were increased in a dose-dependent manner for the males and the high-dose males showed elevated levels of urea, nitrogen and albumin. All anomalies returned to control level after the recovery period.

URINALYSIS
Urine volumes were decreased at all treatment levels in females and specific gravity levels were slightly increased in the high-dose females. These effects are probably due to chance or are related to decreased fee inatke in the high-dose group. No abnormalities were observed at the end of the recovery period.

ORGAN WEIGHTS
- Absolute liver weight and relative liver/body and liver/brain weights were increased for the mid- and high-dose animals in a dose-dependent manner. For the high-dose males, the relative liver/body remained elevated after the recovery period.
- Relative kidney/body weights were slightly increased for the high-dose males and females and for the mid-dose females. Relative kidney/brain weights were slightly increased for the mid- and high-dose females. Absolute kidney weights were slightly decreased in the high-dose recovery animals. The observed kidney effects are most likely a reflection of the decreased body weights.
- The differences in brain weights (viz. decreased absolute and increased relative weights at the high dose) are those expected because of differences in body weight and indicate sparing of the brain during growth inhibition.
- Relative testes weights were increased for the mid- and high-dose males (also in the recovery group) and reflect slightly decreased body weights, rather than target organs effects.

HISTOPATHOLOGY: NON-NEOPLASTIC
Hepatocyte hypertrophy with dose-dependent severity, primarily located in the portal area, was observed in all animals of the high-dose group and in males of the mid-dose group. The hypertrophy was characterized by an increase in the cell size with compression of the adjoining sinusoidal spaces. Furthermore, a decreased incidence of small cytoplasmic vacuoles was observed in a dose-dependent manner. No treatment-related effects were observed in the recovery group.
The minimal hyperplasia of bile ducts, observed in the control and high-dose recovery group, was considered not to be biologically significant.

Effect levels

Dose descriptor:
NOAEL
Effect level:
ca. 300 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Reduced feed consumption and decreased rate of body weight gain

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The liver enlargement was considered to be primarily an adaptive change rather than a toxic effect.

Applicant's summary and conclusion

Conclusions:
The NOAEL after 90 day 2-ethylhexanoic acid containing diet was considered to be 0.5% or ca. 300 mg/kg bw/d due to reduced food consumption and decreased body weight gain.
Based on a read-across approach, the same NOAEL is taken into account in case of sodium 2-ethylhexanoate.
Executive summary:

A read across was performed from the source substance 2 -ethylhexanoic acid to the target substance sodium 2 -ethylhexanoate (for read across justification please refer to attached document, IUCLID Chapter 13).

Groups of 10 male and 10 female ratss were fed diets containing 2 -ethylhexanoic acid at concentrations of 0, 0.1, 0.5 or 1.5% for 90 days. Additional groups of 10 male and 10 female rats were fed either 0 or 1.5% 2 -ethylhexanoic acid diets for 90 days followed by a 28 -day recovery (non-treatment) period. The control diet and the three 2 -ethylhexanoic acid diets provided dose levels of 0, 61, 303 or 917 mg/kg bw/d for males and 0, 71, 360 or 1068 mg/kg bw/d for females. Consumption of 2 -ethylhexanoic acid diets did not result in mortality, significant clinical abnormalities or ophthmlologic 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 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 rcovery 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 blod 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 item involved the liver or metabolic processes associated with the liver. The 0.5 and 1.5% diets were assiciated with increased liver weight and histologic changes including hypertrophied hepatocytes and hepatocyteswith reduced cytoplasmic vacuolization. Cholesterol levels were increased in all dose levels in males, but not in females. Liver effects were reversible upon removal of 2 -ethylhexanoic acid 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 differencee 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 gravitiy for the 1.5% females after 90 -day treatment period. Urine osmolarity 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 collcetion time.

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

The NOAEL after 90 day 2-ethylhexanoic acid containing diet was considered to be 0.5% or ca. 300 mg/kg bw/d due to reduced food consumption and decreased body weight gain.

Based on a read-across approach, the same NOAEL is taken into account in case of sodium 2-ethylhexanoate.