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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Prechronic Toxicity Studies on 2-Ethylhexanol in F334 Rats and B6C3F1 Mice
Author:
Astill B.D. et al.
Year:
1996
Bibliographic source:
Fundamental and Applied Toxicology 29, 31-39

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
Principle of test:

The test substance is orally administered daily in doses of 0, 25, 250 and 500 mg/kg bw/day in 5 male/5 female mice per dosing group for a period of 90 days, 5 days per week. Clinical observations were made, body weights, food consumption. Clinical chemistries, hematologies and selected organ weights were measured, and gross and micropathologies were performed. Target organs were the central nervous system, liver, and forestomach.
Further, 3 mice/sex/dose were used in an ancillary study to determine hepatic peroxisome proliferation. Livers were removed at termination and weighed, and cyanide-insensitive pCoA activities (Lazarow, 1981) and protein concentrations (Lowry et al., 1951) were determined.
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexan-1-ol
EC Number:
203-234-3
EC Name:
2-ethylhexan-1-ol
Cas Number:
104-76-7
Molecular formula:
C8H18O
IUPAC Name:
2-ethylhexan-1-ol
Specific details on test material used for the study:
- Purity: 99.8%

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 43- to 55 days
- Weight at study initiation: 22-29 g (male) and 17-20 g (female)
- Fasting period before study: no
- Housing: were kept singly in plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
castor oil
Remarks:
Cremophor EL
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Doses were prepared daily by dispersing 2-Ethylhexanol in an aqueous solution of Cremophor EL (5 μgl 100 ml) by ultra high speed sonication for 1 min. Homogeneity was maintained by magnetic stirring throughout dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): daily

VEHICLE
- Justification for use and choice of vehicle (if other than water): According to prior studies (Astill et al., 1993) this dose delivery system (oral gavage of an aqueous emulsion of 2-Ethylhexanol by a nonionic surfactant) is provided the most stable dose formulation and minimized gastrointestinal tract irritation and inflammation.
- Concentration in vehicle: 2-Ethylhexanole in an aqueous solution of Cremophor EL (5 μg/100 ml)
- Amount of vehicle (if gavage): Dose volume: 10 ml/kg (based on weekly body weights, controls: 5.0 ml/kg vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations and homogeneity were checked by gas chromatographic analysis of samples from each dose level periodically.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
(+ 3 for ancillary studies)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A high dose of 500 mg/kg/day was selected in the 13-week mouse study because of the findings at 1000 mg/kg/day of female mortality, increased relative liver weight in males, increased relative stomach weight in females, and a 100% incidence of inflammatory forestomach lesions in males and females in the 11-day study.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determined in day -1 and weekly thereafter.

FOOD & WATER CONSUMPTION: Yes
- Food and water consumption were measured weekly

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: Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected from fasted animals on the morning of days 29 and 84.
- Anaesthetic used for blood collection: no
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected from fasted animals on the morning of days 29 and 84.
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
- Body weights
- Organ weights
- Hepatic peroxisome proliferation was determined in an ancillary 13-week study by measuring the activity of hepatic cyanide-insensitive palmitoyl Coenzyme A in livers at termination.
Statistics:
Means and standard deviations were calculated for body weights, food and water consumption, clinical pathology results, and organ weights. Values for test groups were compared with
controls in the main study by ANOVA followed by Dunnen's test (Dunnetl 1955, 1964) and in ancillary studies by ANOVA followed by Student's t test (Winer 1971).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female mouse at 250 mg/kg died during treatment.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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):
There were 14 and 13% increases in relative stomach weight at 250 and 500 mg/kg, respectively, and 6.9 and 5.9% increases in relative liver weight at 250 and 500 mg/kg, respectively. See organ weight changes in the mouse in table 2 in "Any other information on results incl. tables".
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were dark red foci in the glandular stomach of 2/10 females at 500 mg/kg. There were no other gross findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dose-related findings were a moderate focal or multifocal acanthosis in the forestomach of 2/10 males and 1/10 females at 500 mg/kg.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Hepatic peroxisome proliferation:
No increases in pCoA activity were seen in male or female mice at any dose level. See results in table 3 ("Any other information on results incl. tables").
Details on results:
Effects in the 13-week mouse study were limited to increases in relative stomach and liver weight in males at 250 and 500 mg/kg and a low incidence of inflammatory lesions in the forestomach at 500 mg/kg.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 2: Relative Organ Weights in Mice at a Termination in 13 -week Oral Gavage Dosing Studies with 2-ethylhexanol

Males [Dose (mg/kg/day)] Females [Dose (mg/kg/day)]
0 250 500 0 250 500
Mice (n=10)
Liver 3.43 (0.23) 3.82 (0.38)* 4.23 (0.38)** 3.48 (0.34) 3.90 (0.39) 4.16 (0.61)**
Stomach 0.76 (0.09) 0.88 (0.10) 1.03 (0.08)** 0.87 (0.06) 1.03 (0.13)** 1.12 (0 16)**

Values are mean organ/body weight ratios (±SD).

*p< 0.05.

**p< 0.01.

Table 3: Hepatic CN-lnsensitive Palmitoyl Coenzyme A Activities after 13 Weeks of Oral Gavage Dosing of 2-Ethylhexanol

Dose (mg/kg/day)
0 25 125 250 500
Mice (n=3) Mean PCoA activity m nanomol/min/mg of protein (±SD)
Males 4.35 (0.73) 4.76 (0.29) 3.67 (0.42) 3 87 (1.98) 4.33 (1.89)
Females 3.17 ( 1.58) 4.66 (0.82) 5.4 (1.78) 5.31 (1.54) 4.49 (2.27)

*p < 0.001 and **p < 0.05 by ANOVA/Students t test.

Applicant's summary and conclusion

Conclusions:
For the oncogenicity study, 750 mg/kg bw/day was selected as a high dose in the oncogenicity study likely to produce minimal stomach inflammation and yet produce toxicity without seriously affecting the lifespan. 50 mg/kg bw/day was selected as a dose level probably free of treatment-effects.
Executive summary:

In a subchronic toxicity study, 2-ethylhexanol (99.8% purity) was administered to 10 male and 10 female B6C3F1 mice by gavage dose levels of 0, 25, 125, 250 and 500 mg/kg bw/day.

One female mouse died at 250 mg/kg bw but no other mortalities occured during treatment.

No clinical signs and body weight changes were observed as well as hematological and biochemistry findings.

Organ weight changes were observed in the stomach and liver at 250 and 500 mg/kg bw/day.

At 500 mg/kg bw/day dark red foci in the glandular stomach were found. Histopathological findings were multifocal acanthosis in the stomach.

No increases in pCoA activity were seen in male or female mice at any dose level.

Based on these results, the NOAEL is 125 mg/kg bw/day and a high dose level of 750 mg/kg bw/day was selected for an oncogenicity study.

This subchronic toxicity study in the mouse is acceptable and satisfies the principle guideline requirement for a subchronic oral study (OECD 408).