Titanium(4+) ethanolate

Endpoint summary

Description of key information

Repeated toxicity testing was considered unnecessary since this substance undergoes immediate disintegration and there are sufficient data on cleavage product. The intrinsic properties of this substance after repeated administration are related to the main degradation product ethanol. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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.

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

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

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

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

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.

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%).

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.

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.

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles and contains sufficient detail to be able to judge the results reliable as a contribution to the understanding of the repeat dose inhalation toxicity of this substance. Only a single limit dose used in males only and only limited (but important) end points studied, but a NOAEL was established. 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.

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

As part of a study to investigate the inhalation toxicity of an ethanol-gasoline mixture, the toxicological and neurochemical effects of ethanol alone were investigated in male and female rats following a 4-week inhalation exposure, and incorporating a 4 week recovery phase.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks (total of 20 days exposure).

Frequency of treatment:

Rats placed in the inhalation chambers for 6 h/day (7.30am-13.30pm), 5 days/week

Remarks:

Doses / Concentrations:
0 or 6130 ppm ethanol
Basis:
no data

No. of animals per sex per dose:

Ten male and 10 female rats per dose (0 or 6130 ppm ethanol) for main study, and additional groups of 5 male and 5 female rats following treatment were exposed to filtered air for a further 4 weeks (recovery phase) to determine reversibility of effects.

Control animals:

yes

Dose descriptor:

NOAEC

Effect level:

>= 6 130 ppm

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed in male and female rats exposed to 6130 ppm ethanol, other than a number of mild effects regarded as adaptive, and that had generally returned to normal following a 4 week recovery period.

Critical effects observed:

not specified

The investigators reported that only mild, adaptive, effects were seen in ethanol-exposed animals, which had in general returned to normal after cessation of exposure. There was a slight (but statistically significant) increase in relative thymic weight seen in male rats exposed to ethanol (compared to controls), although this had returned to normal after the 4 week recovery phase. Ethanol-exposed female rats at the end of the recovery period showed a slight (but statistically significant) increased relative heart weight, but this was not evident immediately following the 4 -weeks of ethanol exposure. Similarly, treated female rats in the recovery group had a statistically significantly increased serum glucose level, but again this was not apparent immediately following ethanol exposure.

Slight (but statistically significant) neurochemical changes in the brain were reported in female rats exposed to ethanol (compared to controls) [although it is not clear if these were seen immediately following the exposure to ethanol, or were still evident at the end of the 4 -week recovery phase].

There were no treatment-related clinical signs of toxicity and no gross pathological or histological changes were reported on examination of the major organs. Body weights, liver enzyme levels, haematology, and clinical chemistry parameters were otherwise normal.

Conclusions:

Inhalation of ethanol at dose level of 6130 ppm (11 600mg/m3) for 4 weeks produced only mild effects that were regarded as adaptive and returned to normal following a 4 week recovery phase. Based on the study results NOAEC >= 116000mg/m3 was established for males and females.

Executive summary:

As part of a study to investigate the inhalation toxicity of an ethanol-gasoline mixture, groups of 15 male and 15 female rats were exposed in an inhalation chamber to 0 or 6130 ppm (11 600 mg/m3) ethanol, 6 hours/day, 5 days/week, for 4 weeks. Five animals of each sex/group were then exposed to filtered air for a further 4 -weeks recovery period prior to assessment.

Following exposure to ethanol, the investigators reported only mild, adaptive, effects that had generally returned to normal by the end of the recovery period. Ethanol resulted some slight brain neurochemical alterations that were gender-specific, with female rats appearing to be more sensitive than males. The toxicological significance of these changes is not clear.

No clinical signs of toxicity were observed, and there were no gross or microscopic tissue changes seen on examination of a range of major organs (including the heart, liver, spleen, kidney, brain, testes, and thymus).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

11 600 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Acceptably documented study which meets basic scientific principles and contains sufficient detail to be able to judge the results reliable as a contribution to the understanding of the repeat dose inhalation toxicity of this substance. Only a single limit dose used in males only and only limited (but important) end points studied, but a NOAEL was established. 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.

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

As part of a study to investigate the inhalation toxicity of an ethanol-gasoline mixture, the toxicological and neurochemical effects of ethanol alone were investigated in male and female rats following a 4-week inhalation exposure, and incorporating a 4 week recovery phase.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

4 weeks (total of 20 days exposure).

Frequency of treatment:

Rats placed in the inhalation chambers for 6 h/day (7.30am-13.30pm), 5 days/week

Remarks:

Doses / Concentrations:
0 or 6130 ppm ethanol
Basis:
no data

No. of animals per sex per dose:

Ten male and 10 female rats per dose (0 or 6130 ppm ethanol) for main study, and additional groups of 5 male and 5 female rats following treatment were exposed to filtered air for a further 4 weeks (recovery phase) to determine reversibility of effects.

Control animals:

yes

Dose descriptor:

NOAEC

Effect level:

>= 6 130 ppm

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed in male and female rats exposed to 6130 ppm ethanol, other than a number of mild effects regarded as adaptive, and that had generally returned to normal following a 4 week recovery period.

Critical effects observed:

not specified

The investigators reported that only mild, adaptive, effects were seen in ethanol-exposed animals, which had in general returned to normal after cessation of exposure. There was a slight (but statistically significant) increase in relative thymic weight seen in male rats exposed to ethanol (compared to controls), although this had returned to normal after the 4 week recovery phase. Ethanol-exposed female rats at the end of the recovery period showed a slight (but statistically significant) increased relative heart weight, but this was not evident immediately following the 4 -weeks of ethanol exposure. Similarly, treated female rats in the recovery group had a statistically significantly increased serum glucose level, but again this was not apparent immediately following ethanol exposure.

Slight (but statistically significant) neurochemical changes in the brain were reported in female rats exposed to ethanol (compared to controls) [although it is not clear if these were seen immediately following the exposure to ethanol, or were still evident at the end of the 4 -week recovery phase].

There were no treatment-related clinical signs of toxicity and no gross pathological or histological changes were reported on examination of the major organs. Body weights, liver enzyme levels, haematology, and clinical chemistry parameters were otherwise normal.

Conclusions:

Inhalation of ethanol at dose level of 6130 ppm (11 600mg/m3) for 4 weeks produced only mild effects that were regarded as adaptive and returned to normal following a 4 week recovery phase. Based on the study results NOAEC >= 116000mg/m3 was established for males and females.

Executive summary:

As part of a study to investigate the inhalation toxicity of an ethanol-gasoline mixture, groups of 15 male and 15 female rats were exposed in an inhalation chamber to 0 or 6130 ppm (11 600 mg/m3) ethanol, 6 hours/day, 5 days/week, for 4 weeks. Five animals of each sex/group were then exposed to filtered air for a further 4 -weeks recovery period prior to assessment.

Following exposure to ethanol, the investigators reported only mild, adaptive, effects that had generally returned to normal by the end of the recovery period. Ethanol resulted some slight brain neurochemical alterations that were gender-specific, with female rats appearing to be more sensitive than males. The toxicological significance of these changes is not clear.

No clinical signs of toxicity were observed, and there were no gross or microscopic tissue changes seen on examination of a range of major organs (including the heart, liver, spleen, kidney, brain, testes, and thymus).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated oral toxicity

Weight of evidence approach was used to evaluate the testing needs of this endpoint as there are no studies available from the titanium(4+) ethanolate. In order to avoid unnecessary animal testing, further investigations were considered scientifically unjustified since the substance undergoes immediate disintegration and there are sufficiently data on cleavage products.

Titanium(4+) ethanolate hydrolyses rapidly (<5min) when it comes in contact with water or moisture (Brekelmans, M.J.C., 2013). After hydrolysis no significant reaction products other than ethanol and non-hazardous hydrated titanium dioxide (TiO2) exist. Based on the rapid hydrolysis, the intrinsic properties are most likely related to two decomposition products, ethanol and TiO2. Ethanol is the most relevant decomposition product for CSA. TiO2 exists as solid insoluble precipitate having low bioavailability and possessing very low acute and long-term toxicity (US EPA, 1994; WHO, 1982). Thus, it is concluded that no further assessment of TiO2 is needed for repeated dose toxicity.

The effects of ethanol were evaluated in a 90 day sub-chronic repeat dose study, where male rats were given a liquid diet containing ethanol at a level of 1 -10% by weight (Holmberg, 1986). The only significant finding in the 2% dose group was centrilobular steatosis. It is often associated with ethanol consumption but in its mild form is not considered to be a pathological condition. There is evidence from both the glucose dosed animals and the different food intakes from the two different group groups that this condition is more related to the caloric content of ethanol than a substance specific effect. It cannot therefore be considered an adverse effect. Liver yellowing was observed in some of the 3% and 4% dose animals and most of the 5% dose animals. It was considered to be dosage-related. On this basis, the no observed adverse effect level (NOAEL) from this study was 2%, which was approximately equivalent to a dose of 3900mg/kg/day. It should be noted that the study did not conform fully to a guideline study since only male rats used, any haematology or urinalysis and only limited clinical chemistry was performed.

In a well conducted GLP study that closely follows guidelines, rats were exposed to ethanol in drinking water at a level of 5% for a period of 90 days (NTP, National Toxicology Program, 1996). 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 study using distilled water as the vehicle for the urethane, the other 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.

Repeated inhalation toxicity

Weight of evidence approach using the read-across data from the decomposition products was used to evaluate the testing needs as there are no studies available for this substance. In order to avoid unnecessary animal testing, further investigations were considered unjustified since the substance undergoes immediate disintegration and there are sufficient data on cleavage products.

Titanium(4+) ethanolate hydrolyses rapidly (<5min) when it comes in contact with water or moisture (Brekelmans, M.J.C., 2012

). After hydrolysis no significant reaction products other than ethanol and hydrated titanium dioxide exist. Titanium dioxide is the solid insoluble precipitate of this substance. Therefore, inhalation is unlikely route of human exposure of this decomposition product and TiO2 is not further considered in CSA. However, ethanol is volatile decomposition product of titanium(4 +) ethanolate, and therefore the most relevant substance to cause the intrinsic repeated dose toxicity via inhalation.

As part of a study to investigate the inhalation toxicity of an ethanol-gasoline mixture, groups of 15 male and 15 female rats were exposed in an inhalation chamber to 0 or 6130 ppm ethanol, 6 hours/day, 5 days/week, for 4 weeks (Chu, 2005). Five animals of each sex/group were then exposed to filtered air for a further 4–weeks recovery period prior to assessment. Following exposure to ethanol, the investigators reported only mild, adaptive, effects that had generally returned to normal by the end of the recovery period. No clinical signs of toxicity were observed, and there were no gross or microscopic tissue changes seen on examination of a range of major organs (including the heart, liver, spleen, kidney, brain, testes, and thymus). Based on these results the no observed adverse effect concentration (NOAEC) was established to be 6130 ppm (11.6mg/l).

In other study to examine the repeat dose toxicity of ethanol, rats were exposed to a single dose of ethanol vapor at 20mg/l for up to 26 days. Intermediate exposure groups were used to allow changes in clinical chemistry, histopathology and blood ethanol concentrations to be followed with time. The study found a number of transient effects (clinical signs, e.g. lethargy and ataxia, mild hepatic vacuolization and changes to clinical chemistry parameters) but in animals exposed for the full 26 days, the only significant effect noted was an increase in plasma GPT levels, which, in isolation, was not regarded as biologically significant. Thus, the no observed adverse effect level (NOAEL) was established to be 20mg/L (20 000 mg/m³). It was noticeable that the blood ethanol levels in the animals exposed for 26 days were much lower than those exposed for shorter periods indicating pronounced induction of metabolic tolerance.

The above data on inhalation toxicity of ethanol is supplemented by reproductive toxicity data by the inhalation route (Nelson, B.K. et al. 1985); testing up to maximum safe concentration (~50% of the lower explosive limit - 16000ppm), produce no significant adverse effects in a 6 week study. Such conditions would cover all conceivable handling and use scenarios, both normal and abnormal.

Repeated dermal toxicity

No valid study identified for titanium(4+) ethanolate. Testing is not necessary since the substance undergoes immediate disintegration (half-life < 5 minutes and there are sufficient data on cleavage products.

After hydrolysis no significant reaction products other than ethanol and non-hazardous hydrated titanium dioxide exist. As skin contact in use and production of the target substance is not likely and adequate RMMs are in use (see sections 9&10 of CSR), dermal route is not considered relevant route of exposure for the target substance.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No studies available for the target substance which is highly reactive. Data is obtained from the most reliable study performed for the main decomposition product (ethanol) with lowest NOAEL.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No studies available for the target substance which is highly reactive. Data is obtained from the most reliable study performed for the main decomposition product (ethanol) with lowest NOAEC.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No studies available for the target substance which is highly reactive. The study was performed for the main decomposition product (ethanol). No local adverse effects were observed.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Not likely route of exposure.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Not likely route of exposure.

Justification for classification or non-classification

The intrinsic properties of titanium(4+) ethanolate are related to the main degradation product; ethanol. Based on the observations made after the subchronic and subacute ethanol exposures in rats by oral and inhalation routes there is no need for classification of the substance in accordance with the criteria of CLP Regulation 1272/2008 and the EU directive 67/548/EEC.