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Description of key information

In a published 90-day repeat dose oral feeding study in rats using 2 -ethylhexamoic acid, the lowest-observed-adverse-efect level was approximately 300 mg/kg/day in rats primarily based on potential adverse effects on the liver. The NOAEL for male rats was 61 mg/kg/day and the NOEL for female rats was 71 mg/kg/day.

In a published 90-day repeat dose oral feeding study in mice, the lowest-observed-adverse-efect level was approximately 300 mg/kg/day in rats primarily based on potential adverse effects on the liver. NOELs for male and female mice, respectively were 180 and 205 mg/kg/ day. The lowest-observed-adverse-effect level was approximately 1000 mg/kg/day in mice primarily based on potential adverse effects on the liver.

On this basis, the lowest NOAEL of 61 mg/kg/day was assigned to is assigned to TMR (2-hydroxypropyl)trimethylammonium 2-ethylhexanoate).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
2-Ethylhexanoic acid (EHA; CAS no. 149-57-5) was obtained from Eastman Chemical Company (Kingsport, TN, USA). The purity of the test sample was determined to be 99.920.05%, and the mass spectrum was consistent with the structure of EHA.
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Male and female weanling F344 rats were obtained from Charles River Laboratories (Wilmington, MA, USA). Females were nulliparous and non-pregnant.
Animals were observed for approximately 2 wk and determined to be healthy prior to testing.All animals were approximately 6 wk of age at the start of
the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Husbandry and environmental conditions were in compliance with the NIH Guide to the Use and Care of Animals (86-23). Certified feed (Agway1 ProlabTM Animal Diet RMH 3200) and water were available ad lib
Route of administration:
oral: feed
Details on route of administration:
The test substance was added directly (no vehicle) to chow
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
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:
Diets were prepared monthly and the concentrations of test diets were verified analytically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
via Feed ad libitum
Dose / conc.:
0 other: % w/w
Dose / conc.:
0.1 other: % w/w
Dose / conc.:
0.5 other: % w/w
Dose / conc.:
1.5 other: % w/w
No. of animals per sex per dose:
10 males and 10 females
An additional 10 animals per sex were added to the highdose and control groups to evaluate recovery from any observed toxic efects.
Control animals:
yes, plain diet
Positive control:
No
Observations and examinations performed and frequency:
Body weight measurements were collected twice during the first week and at least once weekly thereafter, while feed consumption was usually measured twice weekly throughout the study.
Animals were checked daily for mortality.
Observations for clinical signs of toxicity were performed twice each workday, and 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.
Sacrifice and pathology:
All animals were fasted overnight, anaesthetized with CO2, and exsanguinated from the posterior vena cava after collecting blood for analysis.
Complete gross necropsies were performed on all animals. Haematology and serum clinical chemistries were conducted on five animals per sex.
Other examinations:
Haematology tests included haemoglobin concentration, haematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time.
Clinical chemistry tests included: aspartate aminotransferase, alanine aminotransferase (ALT), total bilirubin, total protein, albumin, creatinine, urea nitrogen, glucose, g-glutamyl transpeptidase, triglycerides, cholesterol, sodium, potassium, chloride,
calcium and phosphorous.
Statistics:
Numerical data were evaluated for statistical significance using the following computer-generated statistical tests: Bartlett’s test (PE0.01), one-way analysis of variance (ANOVA) (PE0.05), and Duncan’s multiple range test (PE0.05).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain was slightly lower for animals consuming diets containing 1.5% EHA compared with the control groups.
Mean body weights of rats consuming diets containing 0.5% or 0.1% EHA were unaffected compared with controls.
Food consumption and compound intake (if feeding study):
effects observed, non-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 dierences 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 specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
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 dierences 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):
Triglyceride concentrations for rats were not significantly different from 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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-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 (PE0.05) than the weights of the respective control groups.
The mean absolute liver weights for all groups fed 1.5% EHA, and for female rats and male mice fed 0.5% EHA were also significantly greater (PE0.05) 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 were unaffected by EHA treatment.
Minor, yet statistically significant (PE0.05) decreases in absolute brain weights (1.5% female rats) not considered biologically significant.
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:
effects observed, non-treatment-related
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, non-treatment-related
Description (incidence and severity):
Hepatocyte hypertrophy was observed after 13 wk of treatment. No hepatic lesions were observed in rats after recovery.
Hepatomegaly, which was noted in the present study, was interpreted as an adaptive response resulting from enzyme induction, particularly since EHA is known to induce enzyme activity in the liver.
No testicular changes were noted.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
61 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
71 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
System:
other: Liver
Organ:
liver

Hepatomegaly, which was noted in the present study, was interpreted as an adaptive response resulting from enzyme induction, particularly since EHA is known to induce enzyme activity in the liver.

Conclusions:
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.
The NOAEL for male rats was 61 mg/kg/day and the NOEL for female rats was 71 mg/kg/day.
The lowest-observed-adverse-efect level was approximately 300 mg/kg/day in rats primarily based on potential adverse effects on the liver. Thus, EHA appears to cause similar hepatic effects
to 2-EH following subchronic exposure, but at higher dose levels.
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no
Specific details on test material used for the study:
2-Ethylhexanoic acid (EHA; CAS no. 149-57-5) was obtained from Eastman Chemical Company (Kingsport, TN, USA). The purity of the test sample was determined to be 99.920.05%, and the mass spectrum was consistent with the structure of EHA.
Species:
mouse
Strain:
B6C3F1
Details on species / strain selection:
B6C3F1 mice were obtained from Charles River Laboratories (Wilmington, MA, USA). Females were nulliparous and non-pregnant.
Animals were observed for approximately 2 wk and determined to be healthy prior to testing.All animals were approximately 6 wk of age at the start of
the study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Husbandry and environmental conditions were in compliance with the NIH Guide to the Use and Care of Animals (86-23). Certified feed (Agway1 ProlabTM Animal Diet RMH 3200) and water were available ad lib
Route of administration:
oral: feed
Details on route of administration:
The test substance was added directly (no vehicle) to chow
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
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:
Diets were prepared monthly and the concentrations of test diets were verified analytically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
via Feed ad libitum
Dose / conc.:
0 other: % w/w
Dose / conc.:
0.1 other: % w/w
Dose / conc.:
0.5 other: % w/w
Dose / conc.:
1.5 other: % w/w
No. of animals per sex per dose:
10 males and 10 females
An additional 10 animals per sex were added to the highdose and control groups to evaluate recovery from any observed toxic efects.
Control animals:
yes, plain diet
Positive control:
No
Observations and examinations performed and frequency:
Body weight measurements were collected twice during the first week and at least once weekly thereafter, while feed consumption was usually measured twice weekly throughout the study.
Animals were checked daily for mortality.
Observations for clinical signs of toxicity were performed twice each workday, and 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.
Sacrifice and pathology:
All animals were fasted overnight, anaesthetized with CO2, and exsanguinated from the posterior vena cava after collecting blood for analysis.
Complete gross necropsies were performed on all animals. Haematology and serum clinical chemistries were conducted on five animals per sex.
Other examinations:
Haematology tests included haemoglobin concentration, haematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time.
Clinical chemistry tests included: aspartate aminotransferase, alanine aminotransferase (ALT), total bilirubin, total protein, albumin, creatinine, urea nitrogen, glucose, g-glutamyl transpeptidase, triglycerides, cholesterol, sodium, potassium, chloride,
calcium and phosphorous.
Statistics:
Numerical data were evaluated for statistical significance using the following computer-generated statistical tests: Bartlett’s test (PE0.01), one-way analysis of variance (ANOVA) (PE0.05), and Duncan’s multiple range test (PE0.05).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight gain was slightly lower for animals consuming diets containing 1.5% EHA compared with the control groups.
Mean body weights of rats consuming diets containing 0.5% or 0.1% EHA were unaffected compared with controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
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 dierences 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):
Triglyceride concentrations were lower (PE0.05) for the 1.5% male mice and for the 0.5 and 1.5% female mice, compared with control values. After recovery, no dierences were seen in female mice, but males still had a 27% lower (PE0.05) 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 (PE0.05) for both 1.5% male and female mice and 0.5% female mice after 13 wk of treatment. Bilirubin levels returned to normal after recovery for female mice, but continued to be lower for male mice.
As hypobilirubinaemia is typically associated with anaemia and no haematological parameters were suggestive of anaemia in rats or mice, these differences may be considered clinically insignificant.
ALT for the 1.5% male mice was higher (PE0.05) at the 13-wk sacrifice. The mean ALT level was lower (PE0.05) after recovery, but this was not considered to be biologically significant.
No haematological differences were seen for mice.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The mean absolute liver weights for all groups fed 1.5% EHA, and for female rats and male mice fed 0.5% EHA were also significantly greater (PE0.05) 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 and mice were unaffected by EHA treatment.
Minor, yet statistically significant (PE0.05) 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.
While slight increases in relative adrenal gland, brain, and testes weights occurred for some of the groups, the dierences were related to reduced terminal body weight and, therefore, were not considered to be indicative of organ specific toxicity.
Gross pathological findings:
effects observed, non-treatment-related
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, non-treatment-related
Description (incidence and severity):
Hepatocyte hypertrophy was observed after 13 wk of treatment, 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.
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 wk. 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 wk.
No testicular changes were noted.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
61 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
71 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
System:
other: Liver
Organ:
liver

Hepatomegaly, which was noted in the present study, was interpreted as an adaptive response resulting from enzyme induction, particularly since EHA is known to induce enzyme activity in the liver.

Conclusions:
The 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.

NOELs for male and female mice, respectively were 180 and 205 mg/kg/ day. The lowest-observed-adverse-effect level was approximately 1000 mg/kg/day in mice primarily based on potential adverse effects on the liver.
Thus, EHA appears to cause similar hepatic effects to 2-EH following subchronic exposure, but at higher dose levels
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
61 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two publshed 90-day feeding studies using 2-EHA in rats and mice were perfomed in equivalence to OECD guidelines and showed similar effects. No GLP although studies are wel documentd and peer reviewed.
System:
hepatobiliary
Organ:
other: Liver

Additional information

Justification for classification or non-classification