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Repeated dose toxicity: oral

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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:
other: GLP-guideline study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1996
Report Date:
1996
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Analytical purity: 99.8% by gas chromatography

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 42-43 days
- Weight at study initiation: 105-114 g (males); 86-97 g (females)
- Fasting period before study: no data
- Housing: singly in stainless steel 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
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 at the start of the preliminary 11 -day studies and periodically in 13-week studies.
Duration of treatment / exposure:
90 days
Frequency of treatment:
5d/week for 13 weeks
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

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected twice daily for morbidity or mortality, or once daily on nontreatment days. Clinical observations were
made daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at start and thereafter at weekly intervals


BODY WEIGHT: Yes
- Time schedule for examinations: at start and thereafter at weekly intervals


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n.a.


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 data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:



HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study days 29 and 84.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: not specified
- Parameters examined: leucocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular
hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential leucocytes, and reticulocytes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on study days 29 and 84.
- Animals fasted: No data
- How many animals: not specified
- Parameters checked in table [1] were examined.


URINALYSIS: No
- Time schedule for collection of urine: n.a.
- Metabolism cages used for collection of urine: No
- Animals fasted: No data



NEUROBEHAVIOURAL EXAMINATION: No data



OTHER:
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:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
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 examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities or clinical findings differing from controls at any treatment level.

BODY WEIGHT AND WEIGHT GAIN
There was decreased weight gain in male and female rats at 500 mg/kg, starting at Week 4 in males and Week 11 in females,
amounting to weight losses of 7% in males and 6% in females at termination (both p<0.01).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
n.a.

FOOD EFFICIENCY
n.a.

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

OPHTHALMOSCOPIC EXAMINATION
n.a.

HAEMATOLOGY
There was a 25% increase in reticulocyte numbers in male and female rats at 500 mg/kg in Week 13.

CLINICAL CHEMISTRY
Differences from controls rats were seen mostly at 84 days (data not shown).
Males at 500 mg/kg/day: 13% decreases in total protein and albumin concentrations.
Females at 250 mg/kg/day: 30 % decrease in serum ALT activities
Females at 500 mg/kg/day: 36% decreases in serum ALT activities;
16% decrease in serum cholesterol concentration

URINALYSIS
n.a.

NEUROBEHAVIOUR
n.a.

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


Table: Relative organ weights (a) in rats at termination of the 13-Week oral gavage rat study (b)

Males [Dose (mg/kg bw/day)] Females [Dose (mg/kg bw/day)]
0 250 500 0 250 500
--------------------------------------------------------------------
Brain 0.68 0.70 0.72** 1.07 1.1 1.1
Kidneys 0.69 0.75** 0.81** 0.77 0.81* 0.82**
Liver 2.77 2.98** 3.57** 2.67 2.88** 3.07**
Stomach 0.57 0.58 0.63** 0.71 0.75* 0.82**
Testes/Ovaries 1.11 1.16 1.17* 0.041 0.037* 0.039
--------------------------------------------------------------------------------------
values are mean organ/body weight ratios
there were no significant differences from controls in rats 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 rats 2/10 males and 4/10 females exhibited single
or multiple slightly elevated foci in the forestomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dose-related findings in rats (data not shown) were limited to the forestomach and liver at 500 mg/kg.
Forestomach: there was a generalized acanthosis of the forestomach mucosa in 1/10 males with ballooning degeneration of the epithelial wall and acanthosis of the forestomach mucosa in 2/10 males and 5/10 females.
Liver: there was a moderate decrease in hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal ß-cell hyperplasia in 3/10 female rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
n.a.

HISTORICAL CONTROL DATA (if applicable)
n.a.

OTHER FINDINGS
Peroxisome proliferation:
In Week 13, increases in pCoA activity were 6.5-fold in male rats (p<0.001) and 3.4-fold in females (p<0.05) at 500 mg/kg . Reportedly, decreases in body weight gain were similar to those in the main study animals.

--------------------------------------------------------------------------------------
Dose (mg/kg bw/day)
0 25 125 250 500
--------------------------------------------------------------------
Mean PCoA activity (nanomol/min/mg of protein)
Males 3.38 4.49 5.21 6.24 22.0*
Females 2.95 4.54 5.01 6.6 9.92**
--------------------------------------------------------------------------------------
*p<0.001 and **p<0.05 by ANOVA/Students ttest

Effect levels

open allclose all
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
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

Any other information on results incl. tables

 

    
 
Table: Relative organ weights (a) in rats at termination of the 13-Week 
oral gavage rat study (b)
    
Males [Dose (mg/kg bw/day)]
Females [Dose (mg/kg bw/day)]
0
250
500
0
250
500
Brain
0.68
0.70
0.72**
1.07
1.1
1.1
Kidneys
0.69
0.75**
0.81**
0.77
0.81*
0.82**
Liver 
2.77
2.98**
3.57**
2.67
2.88**
3.07**
Stomach 
0.57
0.58
0.63**
0.71
0.75*
0.82**
Testes/Ovaries
1.11
1.16
1.17*
0.041
0.037*
0.039
 
(a) values are mean organ/body weight ratios
(b) there were no significant differences from controls in rats at 25 and 125 mg/kg bw/day
* p0.05; ** p0.01    

Applicant's summary and conclusion

Conclusions:
Valid subchronic oral gavage rat study.
(1) 2-EH was moderately toxic at all dose levels up to and including 500 mg/kg bw/day; based
- on lack of mortality and clinical signs;
- moderately reduced body weights in groups at 500 mg/kg bw/day;
- minor effects of low relevance on clinicochemcial and hematological parameters
(2) Target organs were the liver, forestomach, and the kidneys; based on significantly increased relative organ weights at termination
(3) Local irritating effects prevailed; systemic effects were low; based on
- inflammation in the forestomach,
- and lack of treatment-related findings in other organs, including testes
(4) 2-EH induces peroxisome proliferation in rats; observed at 500 mg/kg bw/day in rats of both sexes
(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 rats (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:

  • Mortalities or clinical findings were not different from controls
  • Food consumption was comparable to controls
  • Body weight gain was reduced in the high dose groups, resulting in decreased terminal weights in males (-7%) and females (-6%); (both p<0.01)
  • Clinicochemical changes were seen in high dose groups (males:total protein and albumin -13%; females: serum cholesterol -16%); biological significance is unclear
  • Hematology: reticulocytes were increased (25%) in high dose males and females
  • Organ weights: target organs were liver, forestomach, and kidneys; based on increased relative weights (p<0.01) in male and female groups at 250 and 500 mg/kg bw/day. No weight changes were noted at 25 and 125 mg/kg bw/day.
  • Reproductive organs: relative weight of testes was increased, and that of ovaries decreased, at 250 and 500 mg/kg bw/day.
  • Histopathology revealed changes only in high dose animals. Predominantly inflammatory changes in the forestomach which are attributable to the irritation properties of 2 -EH
  • 2 -EH at 500 mg/kg bw/day caused peroxisome proliferation, as evidenced by a statistically significant increase of the hepatic cyanide-insensitive palmitoyl coenzyme A activity in male (p<0.001) and female (p<0.05) rats in Week 13.
  • In the 25 and 125 mg/kg bw groups no substance related effects occured. The subchronic NOEL was 125 mg/kg bw/day in male and female rats. The estimated NOAEL is 250 mg/kg bw/day.