Titanium(4+) ethanolate

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:

weight of evidence

Study period:

1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Highly reputable testing organisation with detailed report available. Only a single dose, hence only reliable with restrictions, but sufficient to determine a NOAEL. Read-across justification: The substance is hydrolytically unstable. When it comes in contact with water or moisture complete hydrolysis will take place with no significant reaction products other than alcohol and hydrated titanium dioxide. This rapid hydrolysis (hydrolysis half-life < 3 minutes to < 2 hours) is the driving force for the toxicokinetics of target substance. Because of the rapid hydrolysis, the influence of the mode of administration through inhalation, dermal and oral is related to the hazardous degradation product (alcohol) released from the target substance. The identification of degradation products from the hydrolysis study conducted for the target substance verifies that there are no impurities in the alcohol released from the target substance, which might change the hazardous properties of the target substance compared to the properties of the pure alcohol. As there is a mechanistic reasoning to the read-across, the unnecessary animal testing is avoided by using the read-across data from the degradation product (relevant alcohol) to evaluate irritation, sensitization and the short term and long-term toxicological effects and mutagenicity of the target substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

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 level used, no opthalmology

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

Constituent 2

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:

rat

Strain:

Fischer 344

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: 5 per cage.
- Food ad libitum: NIH-07 open formula diet (Zeigler Brothers Inc, Gardeners, PA)
- Water ad libitum

ENVIRONMENTAL CONDITIONS
- 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: 22/24 January 1991 to: 24/26 April 1991; females 21/23 January 1991 to: 23/25 April 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 no treatment

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Assessed at termination and satellite groups on days 3 and 23.
- Number of animals: all
- Parameters examined: hematocrit (Hct), hemoglobin (Hgb) concentration, erythrocyte (RBC) count, reticulocyte count, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), plateletcount, and leukocyte (WBC) count and differential.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Assessed at termination and satellite groups on days 3 and 23.
- Number of animals: all
- Parameters examined: urea nitrogen (UN), creatinine, total protein, albumin, alanine aminotransferase (ALT), alkaline phosphatase, creatine kinase, sorbitol dehydrogenase (SDH), and total bile acids.

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. Clinical chemistry, haematology and 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:

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

ORGAN WEIGHTS
There was a 20% decrease in thymus absolute weight relative to controls (15% relative) for males. However, this was not evident in the parallel series with the urethane in 5% ethanol as a vehicle with some evidence to suggest that the drinking water control was unusually high and the 5% ethanol control unusually low. With only one dose level, dose response cannot be checked and therefore the significance of this result is not clear. With the additional observation that no effect was seen in females, this is not taken as a conclusive adverse effect. In females, there was evidence of a liver weight increase but this was not statistically significant.

HAEMATOLOGY
Reticulocyte count was increased and serum bile acid concentration increased. Some other blood biochemical parameters differed inconsistently from control values at day 3 or 23. Again, there was no evidence of an effect from ethanol exposure in females on the haematology

CLINICAL CHEMISTRY
Serum bile acid concentrations increased in both males and females, but not substantially. The results in males were not consistent from the values at days 3 or 23 (not diferent on day 23). Again, the effect in both sexes was not consistent when comparing urethane dose pairs across the series so the relevance of this finding is not clear.

HISTOPATHOLOGY:
No treatment related effect in males. In females, there was evidence of ethanol causing hepatodiaphragmatic nodules (4/10 animals, none in controls) and kidney nephropathy (4/10 animals, none in controls). Note that kidney nephropathy was seen in all males, including controls

OTHER FINDINGS
Reproductive tissues and sperm counts were not affected by treatment in males but there was evidence for an increase in estrous cycle length in females (15-20%).

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

ca. 3 250 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: no clear biologically significant effects seen. Some marginal effects around this dose level.

2

Dose descriptor:

NOAEL

Effect level:

< 4 400 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: kidney and liver histopathology, estrous cycle length.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Thymus weights (relative (standard deviation)) for males (** and * are highly significant and significant with respect to the concurrent vehicle control.):

Urethane conc (ppm)	0	110	330	1100	3300	10000
Vehicle water	1.04 (0.05)	1.00 (0.03)	0.93 (0.04)	1.00 (0.02)	0.84 (0.03)**	1.00 (0.07)
Vehicle 5% ethanol in water	0.89 (0.03)	0.93 (0.05)	0.87 (0.03)	0.88 (0.03)	0.92 (0.04)	0.76 (0.06)

Retoculocyte count (10^6/ul, standard deviation in paranthesis) at end of 13 week study for males

Urethane conc (ppm)	0	110	330	1100	3300	10000
Vehicle water	0.29 (0.01)	0.28 (0.01)	0.26 (0.02)	0.24 (0.10)	0.27 (0.02)	0.36 (0.03)
Vehicle 5% ethanol in water	0.21 (0.01)	0.27 (0.01)**	0.23 (0.01)*	0.27 (0.02)**	0.25 (0.02)*	0.46 (0.05)**

The comparison between the urethane in water and in 5% ethanol series again suggests that the ethanol has little effect and that the differences between the controls with no urethane may be down to chance. (** and * are highly significant and significant with respect to the concurrent vehicle control.)

Retoculocyte count (10^6/ul, standard deviation in paranthesis) for intermediate sacrifice groups

Time (days	3	23	90
Vehicle water	0.47 (0.02)	0.29 (0.01)	0.29 (0.01)
Vehicle 5% ethanol in water	0.49 (0.02)	0.25 (.0.1)	0.21 (0.01)

For males, the 5% dose was calculated as equivalent to 3250 - 7000 mg/kg body weight over the various urethane dose groups (mean 3500mg/kg), based on average body weight and drinking water consumption. For females, the range is 4400-7000 mg/kg.

Applicant's summary and conclusion

Conclusions:

In a subchronic oral study ethanol was administered in in rats at a level of 5% . Based on the study results following dose descriptors were esteblished: NOAEL for males > 3250mg/kg bw, LOAEL for females 4400 mg kg bw.

Executive summary:

In a well conducted GLP that closely followed guidelines, rats 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 female rats, there were small but clear and significant histopathological changes in the liver (diaphragmatic nodules), accompanied by a non-statistically significant liver weight increase, and an increase in nephropathy (although male rats showed 100% evidence of this in every dose group). Male rats showed an increase in thymus weights, but it was not clear if this was biologically significant and it may have a chance observation. Male rats also showed some slight but inconsistent changes to haematology (reticulocyte count) and clinical chemistry (serum bile acid concentrations), with the latter also seen in females. It was unclear if these changes were biologically significant. A marginal NOAEL of 5% (>3250mg/kg) is selected for males and a LOAEL of 4400mg/kg for females.