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Toxicological information

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
Study period:
1979-03-06 to 1982-04-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This subchronic oral gavage study was conducted using methods comparable to OECD Test Guideline 408 (Repeated Dose 90-day Oral Toxicity Study in Rodents). The study was well documented and adequately conducted. Deviations from the guideline include: 1. only one dose of test substance was used, and the daily dosage was administered in two treatments per day; 2. the total daily dose volume was 20 mL/kg; 3. most, but not all, of the guideline recommended organ weights were conducted; 4. purity of the test substance was not described in the study report.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
, see rationale for reliability above.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ethanol
- Substance type: active
- Physical state: liquid
- Analytical purity: 100% ethanol
- Storage condition of test material: 5 degree C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: around 6 weeks old
- Weight at study initiation: N/A
- Fasting period before study:
- Housing: individually
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 18 to 19 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3 ° F
- Humidity (%): 50 ± 10%
- Air changes (per hr): N/A
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1979-03-06 To: 1979-06-08

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All dosing solutions were prepared fresh weekly with double distilled water. The volumes of dosing solutions were adjusted so that animals in all groups received a total daily dose of 20 mL/kg. Dosage volumes were adjusted once weekly according to the most current body weight.

DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A


VEHICLE
- Justification for use and choice of vehicle (if other than water): N/A
- Concentration in vehicle: 4 mL/kg
- Amount of vehicle (if gavage): 10 mL/kg (per treatment)
- Lot/batch no. (if required): N/A
- Purity: N/A


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the dosing solutions were taken at weekly intervals and shipped to the sponsor for analysis
Duration of treatment / exposure:
7 weeks or 14 weeks
Frequency of treatment:
twice daily, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
4 other: mL/kg
Remarks:
actual ingested
Dose / conc.:
5 other: mL/kg of a mixture containing 16.25% ethanol, water and another substance regarded as inert
Dose / conc.:
10 other: mL/kg of a mixture containing 16.25% ethanol, water and another substance regarded as inert
Dose / conc.:
20 other: mL/kg of a mixture containing 16.25% ethanol, water and another substance regarded as inert
No. of animals per sex per dose:
21
Control animals:
yes, concurrent vehicle
Details on study design:
In this study, five groups of rats (16 or 21 per sex/dose-level) received doses of 0, 5, 10, 20 ml/kg of a mixture containing 16.25% ethanol (5 and 10 ml/kg dose groups were diluted 15 and 10 ml/kg distilled water to make up a dose volume of 20 mg/kg per day) for a period of 7 or 14 weeks. An additional group of rats (21 rats per sex per group) was dosed with 4 ml/kg of USP Ethanol-190proof-100% ethanol diluted with 16 ml/kg distilled water to make up to a dose volume of 20ml/kg/day).

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and prior to necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 7 and week 14
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: week 7 (5 rats/sex/group); week 14 (10 rats/sex/group)
- Parameters checked: erythrocyte count, hemoglobin, hematocrit, leukocyte count, differential leukocyte counts, prothrombin time, acetylcholinesterase

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 7 and week 14
- Animals fasted: Yes
- How many animals: week 7 (5 rats/sex/group from each of the two control groups and the high dose group; week 14 (10 rats/sex/group from all groups)
- Parameters checked were: blood urea nitrogen, glucose, lactate dehydrogenase, creatine phosphokinase, serum glutamate oxalate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, total protein, albumin, globulin

URINALYSIS: Yes
- Time schedule for collection of urine: one day prior to necropsy from all rats scheduled for sacrifice during week 7 and from 10 rats/sex/group during week 14
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked were: appearance, specific gravity, occult blood, protein, pH, bilirubin, ketone, glucose

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes- Lungs, trachea, heart, liver, kidneys, spleen, lymph node, adrenals, thyroids, pituitary, pancreas, bone, bone marrow, marrow smear, testes, epidydimis, prostate, salivary gland, stomach, duodenum, ileum, jejunum, colon, brain, spinal cord, peripheral nerve, eye, skin, mammary gland, gross lesions, tongue, urethra, ureters, uterus, esophagus, ovaries with oviduct

HISTOPATHOLOGY: Yes- Lungs, trachea, heart, liver, kidneys, spleen, lymph node, adrenals, thyroids, pituitary, pancreas, bone, bone marrow, marrow smear, testes, epidydimis, prostate, salivary gland, stomach, duodenum, ileum, jejunum, colon, brain, spinal cord, peripheral nerve, eye, skin, mammary gland, gross lesions, tongue, urethra, ureters, uterus, esophagus, ovaries with oviduct.

Five rats per sex per group randomly chosen from the water control and the ethanol control were sacrificed at week 7. All surviving rats were sacrificed on day 94. The following organs were weighed and organ to body weight ratios were determined on 10 rats per sex per group from all groups: lungs, heart, liver, kidneys, spleen, adrenals, thyroid, pituitary, testes, brain, and ovaries with oviduct.

Histopathological evaluations were performed from 10 rats per sex per group from the water control and pure ethanol groups.
Statistics:
Statistical analyses were performed separately for each sex on hematology and serum biochemistry determination, absolute organ weights, food consumptions and feed efficiencies. For the interim sacrifice control versus treatment group means were compared using the t-test. For the final sacrifice, a one-way analysis of variance (ANOVA) was used to test for overall differences between control and treatment groups. Where the results of the ANOVA tests were found statistically significant, individual treatment versus control group mean comparisons were made using the least significant difference test (LSD). Groups of data failing the Bartlett test for homogenicity of variance were analyzed using the nonparametric Kruskal-Wallis test. Control versus treatment group means were compared using the Wilcoxon rank sum test. The 95 percent level of significance was used in all test procedures.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Occasionally, animals became dyspneic or developed a bloody nasal discharge following gavage but later recovered. These symptoms, which were attributed to aspiration of gavage fluid, occurred more frequently in the ethanol group but were not considered treatment related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rat in the water control and 5 rats in the ethanol treated group died spontaneously or were sacrificed in a moribund condition during the study. Clinical observations and necropsy lesions supported aspiration of gavage fluid or gavage related trauma as being the cause of all these deaths except one (a rat in the ethanol group). Generally, rats in the ethanol group were noted to struggle during gavage, which increased the likelihood of gavage accidents.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No comparison between pure ethanol treated animals and the water control was reported. No statistically significant differences between treatment and control groups were observed in weekly body weights and total body weight gains.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No comparison between pure ethanol treated animals and the control was reported. No statistically significant differences between treatment and control groups were observed.
Food efficiency:
no effects observed
Description (incidence and severity):
No comparison between ethanol treated animals and the water control was reported. No statistically significant differences between treatment and control groups were observed.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No comparison between pure ethanol treated animals and the water control was reported. No statistically significant differences between treatment and control groups were observed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
no ocular changes were seen during the study.
Haematological findings:
no effects observed
Description (incidence and severity):
No statistically significant differences between treatment and control groups were observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
A comparison between pure ethanol treated animals and the water control was not reported. No other significant changes reported.
Urinalysis findings:
not specified
Description (incidence and severity):
No statistically significant differences between treatment and control groups were observed. However, a comparison between pure ethanol treated animals and the water control was not reported.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adrenal weights of female rats were increased by exposure to the ethanol group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All abnormalities occurred at very low frequency and were interpreted to be due directly or indirectly to the method of treatment rather than the test substance.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal focal to multifocal renal tubular epithelial hyperplasia occured at higher frequencies in rats given 20 ml/kg mixture containing ethanol (16.25%) and the 4 ml/kg (100%) ethanol by gavage versus rats given 20 ml/kg water by gavage. It was noted that renal tubular epithelial hyperplasia is a common incidental finding in laboratory rats and it is uncertain whether the higher incidence of this lesion in the ethanol dosed rats compared with water controls is due to a random variation or to ethanol.
Histopathological findings: neoplastic:
not specified

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 730 mg/kg bw/day (actual dose received)
Based on:
other: mixture containing 16.25% ethanol in inert carrier
Sex:
male
Basis for effect level:
other: No effects seen
Remarks on result:
other: dose calculated from amount present in starting solution
Dose descriptor:
LOAEL
Effect level:
3 200 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
3 200 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Any other information on results incl. tables

The purpose of study was to evaluate three doses of compound with ethanol in water used as a control group in addition to water alone. Although some effects of ethanol administration were reported statistical comparison between the ethanol treatment group and the water treatment group was not reported.

Gonadal tissues were examined for both gross and histopathology and no treatment-related effects were detected.

Note that in humans, the kidney is not normally a target organ for toxicity.

Applicant's summary and conclusion

Conclusions:
Oral treatment of rats for 14 weeks with 0, 5, 10 or 20 mg/Kg of a mixture containing 16.25% USP ethanol resulted in dose-related increases significant increases in kidney weights in the mid and high dose groups treated with 20 ml/kg of mixture containing 16.25% USP ethanol or 4ml/kg of 100% ethanol where histopathologic finding revealed increased minimal focal to multifocal renal tubular epithelial hyperplasia in these two treated rats versus the water treated controls. The NOAEL for the study is determined at 10 ml/Kg mixture containing 16.25% ethanol (1.28g/Kg active ethanol) for increased kidney weight and renal tubular epithelial hyperplasia in male rats. The LOAEL for the study is determined at 4 ml/kg for 100% ethanol (3.156 g/Kg active ethanol) for increased kidmey weight and renal tubular epithelial hyperplasia in male rats.
Executive summary:

The purpose of this study was to investigate the toxicity of USP Ethanol -190 proof-100% and a mixture containing 16.25% USP ethanol in Sprague-Dawley rats when administered by oral gavage. A single dose of 4ml/kg of pure ethanol was used and three doses of the diluted ethanol solution (5, 10, 20ml/kg). Individual body weights and feed consumptions were obtained weekly and animals were observed twice daily for clinical signs of toxicity. All animals were examined by slit lamp biomicroscopy and ophthalmoscopy prior to the initiation of treatment and again prior to necropsy. Hematology and clinical chemistry analyses were performed on blood samples taken on week 7 on 10 rats (5 per sex per group) from the water control, 100% USP ethanol control and high dose group of the mixture containing 16.25% ethanol and on week 14 on 20 rats (10 per sex per group) from all groups. Urine samples were collected 1 day prior to necropsy for all rats scheduled for the week 7 sacrifice and from 20 rats (10 per sex per group) on week 14.

Detailed necropsies were performed on all rats in the study. Five rats per sex per group randomly chosen from the water control and the ethanol control were sacrificed at week 7. All surviving rats were sacrificed on day 94. The following organs were weighed and organ to body weight ratios were determined on 10 rats per sex per group from all groups: lungs, heart, liver, kidneys, spleen, adrenals, thyroid, pituitary, testes, brain, and ovaries with oviduct. Histopathological evaluations were performed on 10 rats per sex per group from the water control and ethanol control groups.

 

No statistically significant differences were noted in the weekly body weights and body weight gains between the ethanol mixture treated groups versus the two control groups. Decreased food consumption was observed in the high-dose ethanol mixture males versus the control males during the first half of the study however, these differences between the high-dose ethanol mixture treated group versus the control group were not observed in the second half of the study. No ocular changes were seen in the study. Increased segmented neutrophil counts were noted in the high dose ethanol mixture female group at week 7 however, no treatment-related changes were noted in the hematology data at final sacrifice on week 14.

 

Oral treatment of rats for 14 weeks with 5, 10, 20 ml/kg of mixture containing 16.25% ethanol resulted in dose-related increases in liver to body weight ratios of female rats at final sacrifice. Both mid and high dose females had significantly higher relative liver weights. However, the absolute liver weights of the high dose ethanol treated group, while significantly increased relative to the 100% ethanol treated group, was not different from the water control group. In addition, increased liver weights were observed in the male rats. Significant increases in kidney weights were observed in the mid and high dose groups treated with 10 or 20 ml/kg of mixture containing 16.25% ethanol. Adrenal weights of female rats were increased in both 100% USP ethanol and the 16.25% ethanol mixture control groups. Ovary weights were significant increased at week 7, but were not significantly different from controls by terminal sacrifice.

 

Histopathologic lesions observed in this study were mainly considered common for to the findings of the laboratory for the age and species of rats in the study with exception of increased minimal focal to multifocal renal tubular epithelial hyperplasia in the high dose 20 ml/kg mixture containing 16.25% ethanol and the 100% USP ethanol control treated rats versus the water treated controls. This was considered related to ethanol treatment. It should be noted however that renal tubular epithelial hyperplasia is a common incidental finding in laboratory rats and it is uncertain whether the higher incidence of this lesion in the ethanol dosed rats compared with water controls is due to a random variation or to ethanol. All other lesions in the pulmonary alveoli, glossitis, stomach, pancreas and pericardium were interpreted to be directly or indirectly associated with the method of treatment (gavage) rather than the test substance. Gonadal tissues were examined for both gross pathology and histopathology and no treatment-related effects were detected. 

 

The NOAEL for the study is determined at 10 ml/Kg for a mixture containing 16.25% ethanol for increased kidney weight and renal tubular epithelial hyperplasia in males (equivalent to 1.73g/kg). The LOAEL for this study is determined at 4 ml/kg for 100% USP ethanol (3.16g/kg) for increased kidney weight and renal tubular epithelial hyperplasia in males. Both doses are well above the maximum of 1g/kg widely used as a test ceiling for the maximum dose.