2-ethylhexyl acetate

Repeated dose toxicity: oral

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study; hydrolysis product of 2-ethylhexyl acetate

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1996

Report date:

1996

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Reference 3

Reference Type:

study report

Title:

Unnamed

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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

Details on test material:

- Analytical purity: 99.8% by gas chromatography

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: 49 - 61 days
- Weight at study initiation: 22-29 g (males); 17-20 g (females)
- Fasting period before study: no data
- Housing: singly in plastic cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

containing Chremophor EL (5 µL/100 mL) as emulsifier

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Doses were prepared daily by dispersing 2EH in an aqueous solution of Cremophor EL (5 µg/100 ml) by ultra high speed sonication for 1 min.
Homogeneity was maintained by magnetic stirring throughout dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): a surfactant (Cremophor) was used to facilitate mixing 2-EH with water as 2-EH is insoluble in water. Cremophor EL (Polyoxyl 35 Castor Oil) is a feed additive according to the Regulation of the European Community.
- Concentration in vehicle: 5 µg/100 ml
- Amount of vehicle (if gavage): dose volume was 10 ml/kg bw

Details on analytical verification of doses or concentrations:

Concentrations and homogeneity were checked by gas chromatographic analysis of samples from each dose level at the start of the preliminary 11 -day studies and periodically in 13-week studies.

Duration of treatment / exposure:

90 days

Frequency of treatment:

5/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 125, 250, 500mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of preliminary 11-day studies
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: determination of peroxisome prolieferation (3 animals per dose level; Table [4])
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): random

Positive control:

not required

Examinations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Adrenals, brains, kidneys, livers, stomachs, testes, and ovaries from all animals were weighed, and with other
organs and tissues listed in U.S. EPA Health Effects Guidelines (1987b) fixed in 4% formalin.
HISTOPATHOLOGY: Yes. All tissues from high dose and control animals were stained with hematoxylin—eosin and examined microscopically.
Lungs, livers (including gallbladders in mice), spleens, kidneys, stomachs, sternums, femurs, and femur bone marrows were examined microscopically at intermediate dose levels.
Skin, eyes, female mammary glands, thigh musculatures, and extraorbital lacrymatory glands were not examined in the absence of signs of toxicity.
Livers were also stained with oil red for lipid content and examined microscopically.

Other examinations:

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 (week 13; ancillary group; 3 animals per dose level)

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 Dunnett's test (Dunnett, 1955, 1964)
and in ancillary studies by ANOVA followed by Student's t test (Winer, 1971).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

MORTALITIES AND CLINICAL SIGNS
One female mouse at 250 mg/kg died during treatment; there were no other mortalities or clinical findings differing from controls at any treatment level.

BODY WEIGHT AND WEIGHT GAIN
There were no differences from controls in body weight gain or food consumption in mice (data not shown).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption was not different from that of controls.

FOOD EFFICIENCY
n.a.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
n.a.

OPHTHALMOSCOPIC EXAMINATION
n.a.

HAEMATOLOGY
There were no differences from controls in clinical pathology parameters in treated mice (data not shown).

CLINICAL CHEMISTRY
There were no differences from controls in clinical pathology parameters in treated mice (data not shown).

URINALYSIS
n.a.

NEUROBEHAVIOUR
n.a.

ORGAN WEIGHTS
Relative organ weights: significant differences from control mice were moderate and limited to the liver and the stomach at 250 and 500 mg/kg. Organ weights were increased in both male and female rats (cf. table).


Table: Relative organ weights (a) in mice at termination of the 13-Week oral gavage mouse study (b)
--------------------------------------------------------------------------------------
Males [Dose (mg/kg bw/day)] Females [Dose (mg/kg bw/day)]
0 250 500 0 250 500
--------------------------------------------------------------------
Liver 3.43 3.82* 4.23** 3.48 3.90 4.16**
Stomach 0.76 0.88 1.03** 0.87 1.03** 1.12**
--------------------------------------------------------------------------------------
values are mean organ/body weight ratios
there were no significant differences from controls in groups at 25 and 125 mg/kg bw/day
* p<0.05; ** p<0.01



GROSS PATHOLOGY
Gross lesions differing from controls in both species were seen at 500 mg/kg bw/day only. In the mouse there were dark red foci in the glandular stomach of 2/10 females at 500 mg/kg which may have been incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dose-related findings in mice findings were limited to a moderate focal or multifocal acanthosis in the forestomach of 2/10 males and 1/10 females at 500 mg/kg.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
n.a.

HISTORICAL CONTROL DATA (if applicable)
n.a.

OTHER FINDINGS
Peroxisome proliferation:
The hepatic cyanide-insensitive palmitoyl Coenzyme A activity was not increased at any dose level compared to control mice (table).

--------------------------------------------------------------------------------------
Dose (mg/kg bw/day)
0 25 125 250 500
--------------------------------------------------------------------
Mean PCoA activity (nanomol/min/mg of protein)
Males 4.35 4.76 3.67 3.87 4.33
Females 3.17 4.66 5.40 5.31 4.49
--------------------------------------------------------------------------------------

Effect levels

1

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: absence of treatment-related effects on target organs

2

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: absence of treatment-related systemic effects on target organs

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Valid subchronic oral gavage mouse study.
(1) The toxicity of 2-EH was low at all dose levels up to and including 500 mg/kg bw/day; based on
- lack of mortality and of clinical sign;
- lack of body weight reduction including groups at 500 mg/kg bw/day;
- lack of effects on clinicochemcial and hematological parameters;
(2) Target organs were the liver and the stomach; based on significantly increased relative organ weights at termination
(3) Local irritating effects in the forestomach; low incidence at 500 mg/kg bw
(4) Systemic effects minor; based on lack of treatment-related findings in other organs, including testes
(5) 2-EH did not induce peroxisome proliferation in male and female mice at 500 mg/kg bw/day or below
(5) The NOEL (no observable effect level) was 125 mg/kg bw/day. A NOAEL (no observable adverse effect level) was not derived, but may be estimated to be 250 mg/kg bw/day, based on the above

Executive summary:

The subchronic effects of 2 -EH were studied in a 90-day oral gavage study using male and female B6C3F1 mice (10 animals per sex and dose). The test dose levels were 0, 25, 125, 250, and 500 mg/kg bw/day, based on the results of preliminary 11-day studies.

Key results include:

• there were no mortalities or clinical findings differing from controls
• food consumption was comparable to controls
• body weight gain was comparable to controls
• there were no clinicochemical or hematological changes at any dose level
• Organ weight changes: target organs were liver and forestomach; a dose-related increase of relative weight was seen in males and females; a level of statistical significance (p<0.05) was gained in males at 250 and 500 mg/kg bw/day
• Male and female reproductive organs: no weight change noted
• Histopathology revealed a low incidence of inflammatory changes only in high dose animals; regarded to be incidental; possibly attributable to the irritation properties of 2 -EH
• 2 -EH was not a peroxisome proliferator in B6C3F1 mice at up to and including 500 mg/kg bw/day
• The subchronic NOEL was 125 mg/kg bw/day in male and female rats. The estimated NOAEL is 250 mg/kg bw/day.