Ethanol

Repeated dose toxicity: oral

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: Highly reliable. Single dose but sufficient to determine a NOAEL

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

single dose only used, no opthalmology, clinical chemistry or haematology.

Principles of method if other than guideline:

The study was primarily to assess the toxicity of urethane with ethanol as the vehicle. From the perspective of ethanol, the study involved a test of ethanol and a set of unexposed controls

GLP compliance:

yes

Remarks:

21 CFR part 58

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol

EC Number:

200-578-6

EC Name:

Ethanol

Cas Number:

64-17-5

Molecular formula:

C2H6O

IUPAC Name:

ethanol

Details on test material:

95% ethanol IR.
99.9% pure by GC
Storage at 20C +/-5.

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 - 46 days.
- Source: Taconic Farms, Germantown, NY
- Quarantine: 18-21 days.
- Animals checked for rodent virus antibodies before and after study (and found to be negative.)
- Housing: singley
- Food ad libitum: NIH-07 open formula diet (Zeigler Brothers Inc, Gardeners, PA)
- Water ad libitum

- temperature: 69-75F
- humidity: 35-65%
- Light cycle: 12 hours dark/12 hours light
- air changes: 10 per hour minimum

IN-LIFE DATES: From: Males: 10/12th December 1990 to: 12/14th March 1991. Females: 11/13th December 1990 to: 13/15th March 1991

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Ethanol was diluted in deionized water. Storage at 4C +/-3 for max of 3 weeks before renewal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of dosing solutions checked throughout study and found to be within 10% of nominal concentration at all times.

Duration of treatment / exposure:

90 days

Frequency of treatment:

7 days/week ad libitum

Doses / concentrations

Remarks:

Doses / Concentrations:
5% w/v in deionized water
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for selecting satellite groups: Satellite animals were included for haematological and clinical chemistry examination at 3 and 23 days.
- Doses were based on literature reports

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly

Sacrifice and pathology:

Complete necropsies performed on all animals
GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)
Organs wieghed: heart, right kidney, liver, lungs, right testis, and thymus.
Histopathologic Examination: adrenal glands, brain (three sections), clitoral glands, esophagus, eyes (if grossly abnormal), femur and marrow, gallbladder (mice only), gross lesions and tissue masses, heart, kidneys, large intestine (cecum, colon, rectum), liver (two sections), lungs, lymph nodes (mandibular and mesenteric), mammary gland, nasal cavity and turbinates (three sections), ovaries, pancreas, parathyroid glands, pituitary gland, preputial glands, prostate gland, salivary gland, seminal vesicle, small intestine (duodenum, jejunum, ileum), spinal cord and sciatic nerve (if neurologic signs were present), spleen, stomach (forestomach and glandular stomach), testes (with epididymis), thigh muscle (if neuromuscular signs were present), thymus, thyroid gland, trachea, urinary bladde,r uterus, and vagina (females in vaginal cytology studies only).

Other examinations:

Sperm motility was assessed at termination. Source used left epididymis. Parameters examined: sperm motility, density and spermatid head count.
Vaginal cytology was assessed for all females of each group by daily (final 12 days) from vaginal smear and cell staining. Relative number of leukocytes, mucleated epithelial cells and large squamous epithelial cells uded to identify estrous stage.

Statistics:

Statistical tests were t-tests and F-tests.
Organ and body weight: parametric multiple comparisons procedures of Williams or Dunnett. Sperm analysis: Non parametric analysis method of Shirley or Dunn.
Jonckheere's test to assess significance of dose response trends and whether a trend sensitive test (William's or Shirley's test was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose response (Dunnett's or Dunn's test).
Significance tested at p<0.05 and p<0.01.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

ORGAN WEIGHTS
Males: relative and absolute liver weight was increased and there were increases in absoluted heart, liver, kidney and lung weight. No effects in females.

HISTOPATHOLOGY
Males: Minimal nephropathy occurred in 30% of treated animals and in 10% controls which may be biologically but is not statistically significant. Females: Minimal nephropathy occurred in 1 treated animals but not in controls which may be biologically but is not statistically significant. However, this finding is not regarded as conclusive

OTHER: Sperm count in the cauda epididymis was decreased (~30%). No effects on estrous cycle length. The only treatment-related change in female mice was a small change to the time spent in dioestrus and pro-oestrus but it was unclear whether this was significant. Cycle length was not significantly changed.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

< 9 700 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: organ weight changes, decreased sperm count, marginal nephropathy

2

Dose descriptor:

NOAEL

Effect level:

> 9 400 mg/kg bw (total dose)

Sex:

female

Basis for effect level:

other: no clear significant biological changes seen

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

In a well conducted GLP study that closely followed guidelines, mice were dosed exposed to ethanol in drinking water at a level of 5% for a period of 90 days. Only a single dose level was used as the study was primarily looking at the toxicology of urethane. To establish the effect of ethanol on urethane disposition, two parallel studies were run, one using distilled water as the vehicle for the urethane, the second using 5% ethanol solution as the vehicle. The study allowed a comparison of the two vehicles used. In male mice, relative and absolute liver weight was increased and there were increases in absoluted heart, liver, kidney and lung weight. There was some evidence for a marginal increase in nephropathy in male mice, but the increase was not statistically clear. Sperm count in the cauda epididymis was also decreased (~30%). Female mice showed no effects apart from a small change to the time spent in dioestrus and pro-oestrus but it was unclear whether this was either statisticall or biologically significant. Cycle length was not significantly changed. A marginal NOAEL of >5% (>9400mg/kg) is selected for females and a LOAEL of 9700mg/kg for males.