Tetra-n-butyl titanate, polymer with water

Endpoint summary

Description of key information

Repeated toxicity testing was considered unnecessary since the target substance undergoes immediate disintegration and there are sufficient data on cleavage product for the oral and inhalation exposure routes. The intrinsic properties of this substance after repeated administration are related to the most hazardous degradation product, n-butanol.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

August 19, 1985 to November 26, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

The study was conducted in accordance with standard operating procedure of Toxicity Research Laboratory (TRL) with Compliance of GLP but do not specify guideline followed.

Qualifier:

no guideline available

Principles of method if other than guideline:

Four groups of male and female Sprague-Dawley rats (30/sex/group) were administered daily 0, 30, 125 or 500 mg/kg bw/day for either 6 or 13 weeks.

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charls River Breeding laboratory, Inc., Portage, Michigan
- Age at study initiation: 22-23 days
- Weight at study initiation: 45-55 gram
- Diet (e.g. ad libitum): Purina certified Rodent laboratory chow #5002 (pellet)
- Water (e.g. ad libitum): Filtered municple water, analyzer periodically for the presence of contaminants as defined by EPA.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70.2 ± 2.2
- Humidity (%): 47.6±9.2
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 12, 1985 To: november 25 and 26, 1985

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance analysis was done by Gas chromatography -Hewlett Packard 5880 with automated sampler and varian flame ionization detector is used with following standard set
Injector Temperature : 150 ̊C
column Temperature : 140 ̊C isothermal
carrier flow : 25 ml/main N2
injection volume: 2 µl

Duration of treatment / exposure:

6 weeks exposure for 10 animals per group for interim sacrifice and 13 weeks exposure for final sacrifice.

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 30, 125, 500 mg/kg/day
Basis:
no data

No. of animals per sex per dose:

30 animals per sex per dose in four group and 10 animals per sex per dose for fifth group

Control animals:

yes

Details on study design:

The amounts administered were based the most recent individual weekly body weight values.
N-Butanol Animals number (sacrifice)
Group Dose(mg/kg /day) male Female
interim final interim final
I 0 10 20 10 20
II 30 10 20 10 20
III 125 10 20 10 20
IV 500 10 20 10 20
V Base Line 10 20 10 20

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
Cage side observations include clinical in File 1( attached below) were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: Yes / No / No data

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 / No / No data

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pretreatment and on week 13
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to treatment and on the time of nacropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: No data
- How many animals: 10 animals per sex per dose group
- Parameters checked in File 2 (attached below) were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment from dose group V and on the time of nacropsy
- Animals fasted: No data
- How many animals: 10 animals per sex per dose group


URINALYSIS: Yes
- Time schedule for collection of urine: prior to treatment from dose group V and 3-5 days prior to sacrifice
- Metabolism cages used for collection of urine: Yes, for 4 hours
- Animals fasted: No data



OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Statistics:

The body weight, food consumption, clinicopathologic, and organ weight data were tested for homogeneity of variance by Bartlett's method. If data were found to be homogeneous difference between control and treatment means were tested for statistical significance by method of Dunnett.If data not found to be homogeneous, The method of Gill (modified Dunnett's ) was employed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment related ataxia first appeared in the high dose group during week 8. Ataxia and hyperactivity occurred in frequently during weeks 9 and 10. These sign increased to a weekly incidence of 32% and 29% for atxia and hypoactivity respectively. On set of ataxia and hyperactivity was about 2-3 minutes after dosing and duration was less then one hour.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment related ataxia first appeared in the high dose group during week 8. Ataxia and hyperactivity occurred in frequently during weeks 9 and 10. These sign increased to a weekly incidence of 32% and 29% for atxia and hypoactivity respectively. On set of ataxia and hyperactivity was about 2-3 minutes after dosing and duration was less then one hour.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment related effect was present on body weight and weight gain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No treatment related effect was present on food consumption. Statistically significant difference between control and treated group and food consumption would occur sporadically,but no trend was observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Total Lesions 1, 2, 4, 5 ( including chorioretinal hypoplasia) were seen as in Control, Low dose, mid dose, high dose, respectively.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At interim sacrifice, RBC(p≤ .05), PCV and HCB,(p ≤.01) in high dose female were 5% less than thr control group. The RBC and PCV (p≤ .05) average for middle dose were also slightly ( 4% and 3% respectively) than control over ages.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At interim sacrifice, lower cholesterol level (p≤ .05) in high dose males, higher neutrophill count (p≤ .05) in mid dose male and at final evaluation lower lymphocyte count(p≤ .05) , higher neutrophill count in low dose females were observed.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Higher (p≤ .05) urine pH values in the low dose males at the interim evaluation and in low dose females at the final evaluation were observed.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No apparent treatment related effect on organ weight value. The only statistically significant difference between control and high dose males was slightly(p≤ .05) higher thyroid weight. No dose response relationship was present.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment related lesion was observed in gross necropsy at the interim or final sacrifices or of the rats found dead or sacrificed in extremes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

No treatment related lesion was observed at histopathologic evaluation. The diffuse subacute lymphadenitis of mendibular lymph node was visible grossly as red or enlarged lymph nodes. This is a commonly observed lesion in laboratory rats.

Histopathological findings: neoplastic:

no effects observed

Details on results:

At interim evaluation, RBC, PCV, and HGB averages in the high dose females were 5% below control averages. The difference were statistically significant but biologically significance is questionable.
HISTOPATHOLOGY: NON-NEOPLASTIC . - Parameters checked in File 4 (attached below) were showing incidence of lesion examined.
BODY WEIGHT AND WEIGHT GAIN: change in mean organ weight were examine as shown in file 3(attached below)

Key result

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

Critical effects observed:

not specified

Read-across justifications and data matrices are presented in IUCLID section 13.

Conclusions:

Oral administration of normal butanol at 500 mg/kg/day produced ataxia and hyperactivity at maximum weekly incidence rate of 32% and 29% respectively. A slightly lower (compare to controls) RBC, PCV and HGB concentration present in the 500 mg/kg/day dose group females at the interim evaluation only may have been treatment related. No treatment related effect were observed at 30 mg/kg/day and 125 mg/kg/day.

Executive summary:

Based on the study results NOAEL was concluded to be 125 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

125 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic 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:

Comparable to guideline study. 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:

according to guideline

Guideline:

EPA OTS 798.2450 (90-Day Inhalation Toxicity)

Deviations:

yes

Remarks:

the tissues from the central and peripheral nervous systems were not examined histologically

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Kingston, USA
- Age at study initiation: ca. 60 d
- Mean weight at study initiation: 271+-7 g (males); 215+-8 g (females); the variability in body weight of individual animals in the selected population did not exceed 20% of the mean for each sex
- Housing: individually in stainless-steel, wire-mesh cages
- Diet: Certified Rodent Diet ad libitum
- Water: filtered municipal tap water

ENVIRONMENTAL CONDITIONS
not reported

Route of administration:

inhalation: vapour

Vehicle:

air

Details on inhalation exposure:

The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The distillation columns were heated to approximately 50°C to enhance vaporization. The oxygen content of the chamber exposure atmosphere was at least 19.0%. The total airflow was set at 12–14 air changes per h. The temperature and humidity were maintained at 20.6–24.7°C and 36.7–68.7%, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapor concentrations were determined at least once each hour with an infrared gas analyzer set at a wavelength of 3.38 µm.

Duration of treatment / exposure:

6 h

Frequency of treatment:

5d/week = 65 exposure days within 13 weeks

Remarks:

Doses / Concentrations:
ca. 2.35, 7.05 and 14.1 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
500, 1500 and 3000 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A concentration of 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration (based on the results of a 2-week repeated exposure study in which animals were exposed to 0, 750, 1500 or 3000 ppm nBA).

Observations and examinations performed and frequency:

OBSERVATIONS
Body weights and feed consumption were measured weekly prior to exposure. Animals were fasted the day prior to necropsy. Fasted body weights were measured after exsanguination, but prior to necropsy. This procedure is standard practice in the laboratory because it allows for blood collection from the posterior vena cava while the animal is under anesthesia, and reduces the variability in the ratio of body and organ weight due to differences in body fluids. Before and after exposure, each rat was removed from its cage and examined. Cageside observations were conducted once a day on weekends. Observations included, but were not limited to, examination of the hair, skin, eyes and mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns.

HEMATOLOGY and CLINICAL CHEMISTRY
Animals were fasted beginning after their last exposure. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The blood was placed into vacutainer tubes and allowed to clot for analyses of serum. Other tubes containing an anticoagulant were used for analyses of whole blood samples. Blood smears were also prepared for blood cell counts. Following blood collection, the animals were killed humanely by exsanguination under anesthesia. Animals were bled and euthanatized in random order based on a computer-generated list.
Whole blood samples were analyzed for red blood cell count, total white blood cell counts, hemoglobin, hematocrit and red blood cell indices using an hematology analyzer. Prothrombin time was measured using a Fibrosystems analyzer. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count.
Serum samples were analyzed for total protein, total bilirubin, calcium, phosphorous, urea nitrogen, creatinine, glucose, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and alkaline phosphatase using a serum analyzer. Albumin concentration and isozyme profile were determined using a Gel Electrophoresis System. The albumin/globulin ratio was calculated from total protein and albumin concentrations. Serum sodium and potassium concentrations were determined using a Photometer and serum chloride concentration was measured using a chloride Analyzer.

OPHTHALMOSCOPY
All rats were examined by a veterinarian for retinal and corneal lesions prior to the start of the study using a direct ophthalmoscope. During the last week of exposure, animals from the control and high-concentration groups were re-examined. Because no changes were detected in the eyes of the high-concentration animals, the animals from the low- and mid-concentration groups were not re-examined.

Sacrifice and pathology:

After 13 weeks of exposure, animals were fasted overnight. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The animals were then exsanguinated and weighed. Wet weights of the liver, kidneys, testes or ovaries, spleen, adrenal glands, lungs and brain were recorded for all animals at necropsy. Paired organs were weighed together, except for the testes, which were weighed individually. All tissues listed in the US EPA Health Effects Testing Guideline for Inhalation Toxicity (40 CFR 798.2450) were collected and preserved in 10% buffered formalin (pH 7.4). The capsule of the right testis was pricked with a 22-gauge needle and preserved in Millonig’s fixative (10% neutral buffered formalin with phosphate buffered saline, pH 7.4).
All tissues listed below were embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (H&E). The nasal passages were decalcified prior to being embedded and sectioned. The lungs were sectioned along a plane allowing visual examination of the major bronchi and bronchioles. The right testis was embedded in glycol methacrylate, sectioned, and stained with H&E and periodic Schiff’s reagent. All tissues except for the brain, spinal cord and peripheral nerve were examined microscopically from the control and high-concentration groups. Nervous tissue from animals exposed simultaneously was evaluated in the neurotoxicity study (CMA 1996b). The lungs, nasal passages, thymus (males only), stomach (females only) and gross lesions were examined from the mid- and low concentration groups.

Other examinations:

The left testis and left epididymis of each male rat were placed into individual bags and frozen at -25°C for sperm counts. Samples were shipped to Research Triangle Institute (Research Triangle Park, NC, USA) for analysis. The frozen tissues were weighed to assess the effect of freezing, and homogenized according to the procedure described in Fail et al. (1991). The number of elongated spermatids (testes) or spermatozoa (epididymis) were counted. The results are expressed per gram tissue weight.

Statistics:

Body weight, feed consumption, serum chemistry, hematology, organ weight and sperm count data were evaluated using the following statistical tests: Bartlett’s test (P<=0.01), one-way analysis of variance (ANOVA) (P<=0.05), and Duncan’s multiple range test (P<=0.05) or Dunnett’s test to indicate statistical significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.
A probability of P<=0.05 (two-tailed) was used to determine significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.

Details on results:

MORTALITY
No mortality occurred in any of the treated groups during the study.

CLINICAL SIGNS
Animals exposed to 3000 ppm (ca. 14.1 mg/L) had reduced activity levels during exposure that were of generally minor severity. Reduced activity was defined as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. Signs of diarrhea and red discoloration on the chin hair were also observed. Animals exposed to 1500 ppm (ca. 7.05 mg/L) appeared normal for the first 5 h of day 0 and the first hour or two of days 1 and 2, then exhibited reduced activity of generally minimal severity for the remainder of the daily exposure periods. Reduced activity of minimal severity was generally seen throughout daily exposures thereafter. Control and 500 ppm (2.350 mg/L) animals appeared normal during exposure.

BODY WEIGHT and FOOD CONSUMPTION
Body weights for the 1500 and 3000 ppm groups (ca. 7.05 and 14.1 mg/L) were significantly lower than the control group for most of the study. Overall weight gains for the 3000 ppm group were 62 and 78% of weight gains for the control group (males and females, respectively), while overall weight gains for the 1500 ppm groups were 77 and 70% of the control group (males and females, respectively). Feed consumption for the 3000 ppm groups were significantly lower than for the control group throughout the study for male rats and at all intervals except days 84 and 91 for female rats. Mean feed consumption values for the 1500 ppm groups were significantly lower than the control group for the majority of the study, and sporadically lower for the 500 ppm groups compared with the control group.

CLINICAL CHEMISTRY and HEMATOLOGY
No effects considered as biologically relevant were observed.
No significant differences in hematologic parameters were seen after 30 days on test. Significantly higher mean erythrocyte counts, hemoglobin concentration and hematocrit values were observed for the 3000 ppm male and female rats after 90 days on test compared with the control groups . The mean eosinophil percentage formale 3000 ppm rats was also significantly higher than for the control group. All the values were within normal limits for rats of this age and for the age and strain of the animals used in this laboratory, and none of the differences were considered biologically significant.
After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower than for the control group. The differences were slight (ca. 1 Meq/L), however. The mean chloride concentration for the 1500 ppm male group was significantly lower compared with the control group. However, this difference was also small (< 4 Meq/L). No other differences in serum chemistries were seen among groups. After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower than for the control group. Mean sorbitol dehydrogenase activity for the 1500 ppm male group was significantly higher than for the control group. These changes were not considered to be toxicologically meaningful, and no other differences in serum chemistry were observed among groups.

OPHTHALMOSCOPY
No treatment-related ophthalmologic changes were observed.

ORGAN WEIGHTS
Terminal body weights measured after exsanguination were significantly lower for the 1500 and 3000 ppm male and female groups compared with the control group. Absolute weights of the liver, kidneys and spleen reflect this reduced body weight. Liver and spleen weights for the 1500 and 3000 ppm male and female groups were significantly lower than for the control groups. Absolute kidney weights for the 1500 ppm female and 3000 ppm male and female groups were also significantly lower than for the control groups. However, relative organ weights (to body weight) for these organs were not significantly different with the exception of the spleen-to-body weight ratio for the 3000 ppm male group, which was significantly lower than for the control group.
Reduced body weight was also reflected in the significantly lower absolute brain weight for the 3000 ppm male group and significantly higher brain-to-body weight for the 1500 ppm female and 3000 ppm male groups compared with their respective control groups. In addition, testes-to-body weights for the 1500 and 3000 ppm male groups and the relative lung (to body weight) weight for the 3000 ppm male group were significantly higher than for the control group. Adrenal gland-to-body weight ratios for the 1500 ppm female and 3000 ppm male and female groups were significantly higher than for the respective control groups.

NECROPSY
Exposure-related changes were observed in the nasal passages and stomachs of 1500 and 3000 ppm rats. All male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium. Degeneration of the olfactory epithelium was observed along the dorsal medial meatus. Degeneration and regeneration of the epithelium was seen along the third ethmoturbinate and several others. The lesion was characterized by karyorrhexis, pyknosis and depletion of olfactory epithelium cells. The severity of the olfactory lesion was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. Olfactory epithelium was replace in some areas by transitional or respiratory epithelium. A few (3/10) 3000 ppm female rats had acute inflammation and degenerative lesions (erosion) of the stomach mucosa (glandular vs forestomach). The severity was minimal to mild.
Lesions of this type in the stomach may be associated with swallowing of mucous containing the test substance, but due to the location of the lesion are more likely caused by stress (Glavin et al., 1991: The neurobiology of stress ulcers. Brain Research Reviews 16, 301–343). An occasional 3000 ppm male rat had atrophy of the thymus, but this was not considered to be a direct compoundrelated effect. Instead, this lesion was attributed to stress (Greaves and Faccini, 1992: Rat Histopathology, A Glossary for Use in Toxicity and Carcinogenicity Studies. p. 51. Elsevier Science, Amsterdam).

SPERM COUNTS
No dose-related or statistically significant effect on epidydimidal or testicular sperm count was observed compared with controls, although the epididymidal sperm counts for all treated groups were lower than controls.

Dose descriptor:

NOAEL

Remarks:

local and systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 500 ppm; based on reduced body weight, food consumption and transient CNS effects; signs of necropsy of the olfactory epithelium

Critical effects observed:

not specified

Read-across justifications and data matrices are presented in IUCLID section 13.

Conclusions:

The repeated dose inhalation toxicity study conducted using n-butyl acetate, the metabolic precursor of n-butanol, provides surrogate data for n-butanol. The NOAEL for n-butyl acetate is considered to be 2.35 mg/l. This NOAEL corresponds to 1.52 mg n-butanol /l (corrected for molecular weight).

Executive summary:

The repeated dose inhalation toxicity study conducted using n-butyl acetate, the metabolic precursor of n-butanol, provides surrogate data for n-butanol. Rats (15/sex/dose level) were exposed to vapor concentrations of 0, 2.35, 7.05, 14.1 mg/l n-butyl acetate (i.e. 0, 500, 1500 and 3000 ppm) for 6 hours per workday during 13 weeks. Rats exposed to 7.05 mg/l and 14.1mg/l n-butyl acetate for 13 weeks decreased body weight, decreased weights of certain organ weights, decreased feed consumption were observed. However, no systemic, organ-specific toxicity was observed. Minimal, transient narcosis and sedation effects were also observed in rats exposed to 7.05 mg/l and 14.1 mg/l n-butyl acetate during exposure. No cumulative effect on activity during the 13-week exposure was observed. Thus, the NOAEL for n-butyl acetate is considered to be 2.35 mg/l. The NOAEL of 2.35 mg/l for n-butyl acetate corresponds to 1.52 mg n-butanol /l (corrected for molecular weight).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 520 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic 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:

Comparable to guideline study. 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:

according to guideline

Guideline:

EPA OTS 798.2450 (90-Day Inhalation Toxicity)

Deviations:

yes

Remarks:

the tissues from the central and peripheral nervous systems were not examined histologically

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Kingston, USA
- Age at study initiation: ca. 60 d
- Mean weight at study initiation: 271+-7 g (males); 215+-8 g (females); the variability in body weight of individual animals in the selected population did not exceed 20% of the mean for each sex
- Housing: individually in stainless-steel, wire-mesh cages
- Diet: Certified Rodent Diet ad libitum
- Water: filtered municipal tap water

ENVIRONMENTAL CONDITIONS
not reported

Route of administration:

inhalation: vapour

Vehicle:

air

Details on inhalation exposure:

The test atmosphere was generated by metering the test substance into glass distillation columns packed with glass beads. Filtered, compressed air was passed through the glass bead-packed columns to evaporate the test substance. The distillation columns were heated to approximately 50°C to enhance vaporization. The oxygen content of the chamber exposure atmosphere was at least 19.0%. The total airflow was set at 12–14 air changes per h. The temperature and humidity were maintained at 20.6–24.7°C and 36.7–68.7%, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber vapor concentrations were determined at least once each hour with an infrared gas analyzer set at a wavelength of 3.38 µm.

Duration of treatment / exposure:

6 h

Frequency of treatment:

5d/week = 65 exposure days within 13 weeks

Remarks:

Doses / Concentrations:
ca. 2.35, 7.05 and 14.1 mg/L
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
500, 1500 and 3000 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A concentration of 3000 ppm was selected as an exposure concentration that would produce overt signs of toxicity, and 500 ppm was selected as an exposure concentration that was expected to have no effect. An exposure concentration of 1500 ppm was selected as the intermediate exposure concentration (based on the results of a 2-week repeated exposure study in which animals were exposed to 0, 750, 1500 or 3000 ppm nBA).

Observations and examinations performed and frequency:

OBSERVATIONS
Body weights and feed consumption were measured weekly prior to exposure. Animals were fasted the day prior to necropsy. Fasted body weights were measured after exsanguination, but prior to necropsy. This procedure is standard practice in the laboratory because it allows for blood collection from the posterior vena cava while the animal is under anesthesia, and reduces the variability in the ratio of body and organ weight due to differences in body fluids. Before and after exposure, each rat was removed from its cage and examined. Cageside observations were conducted once a day on weekends. Observations included, but were not limited to, examination of the hair, skin, eyes and mucous membranes, motor activity, feces, urine, respiratory system, circulatory system, autonomic nervous system, central nervous system, and behavior patterns.

HEMATOLOGY and CLINICAL CHEMISTRY
Animals were fasted beginning after their last exposure. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The blood was placed into vacutainer tubes and allowed to clot for analyses of serum. Other tubes containing an anticoagulant were used for analyses of whole blood samples. Blood smears were also prepared for blood cell counts. Following blood collection, the animals were killed humanely by exsanguination under anesthesia. Animals were bled and euthanatized in random order based on a computer-generated list.
Whole blood samples were analyzed for red blood cell count, total white blood cell counts, hemoglobin, hematocrit and red blood cell indices using an hematology analyzer. Prothrombin time was measured using a Fibrosystems analyzer. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count. Slides with blood smears were stained and examined for cellular morphology, differential white blood cell count and platelet count.
Serum samples were analyzed for total protein, total bilirubin, calcium, phosphorous, urea nitrogen, creatinine, glucose, gamma-glutamyltransferase, aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase and alkaline phosphatase using a serum analyzer. Albumin concentration and isozyme profile were determined using a Gel Electrophoresis System. The albumin/globulin ratio was calculated from total protein and albumin concentrations. Serum sodium and potassium concentrations were determined using a Photometer and serum chloride concentration was measured using a chloride Analyzer.

OPHTHALMOSCOPY
All rats were examined by a veterinarian for retinal and corneal lesions prior to the start of the study using a direct ophthalmoscope. During the last week of exposure, animals from the control and high-concentration groups were re-examined. Because no changes were detected in the eyes of the high-concentration animals, the animals from the low- and mid-concentration groups were not re-examined.

Sacrifice and pathology:

After 13 weeks of exposure, animals were fasted overnight. The following day, animals were anesthetized with Metofane, and blood was collected from the posterior vena cava. The animals were then exsanguinated and weighed. Wet weights of the liver, kidneys, testes or ovaries, spleen, adrenal glands, lungs and brain were recorded for all animals at necropsy. Paired organs were weighed together, except for the testes, which were weighed individually. All tissues listed in the US EPA Health Effects Testing Guideline for Inhalation Toxicity (40 CFR 798.2450) were collected and preserved in 10% buffered formalin (pH 7.4). The capsule of the right testis was pricked with a 22-gauge needle and preserved in Millonig’s fixative (10% neutral buffered formalin with phosphate buffered saline, pH 7.4).
All tissues listed below were embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin (H&E). The nasal passages were decalcified prior to being embedded and sectioned. The lungs were sectioned along a plane allowing visual examination of the major bronchi and bronchioles. The right testis was embedded in glycol methacrylate, sectioned, and stained with H&E and periodic Schiff’s reagent. All tissues except for the brain, spinal cord and peripheral nerve were examined microscopically from the control and high-concentration groups. Nervous tissue from animals exposed simultaneously was evaluated in the neurotoxicity study (CMA 1996b). The lungs, nasal passages, thymus (males only), stomach (females only) and gross lesions were examined from the mid- and low concentration groups.

Other examinations:

The left testis and left epididymis of each male rat were placed into individual bags and frozen at -25°C for sperm counts. Samples were shipped to Research Triangle Institute (Research Triangle Park, NC, USA) for analysis. The frozen tissues were weighed to assess the effect of freezing, and homogenized according to the procedure described in Fail et al. (1991). The number of elongated spermatids (testes) or spermatozoa (epididymis) were counted. The results are expressed per gram tissue weight.

Statistics:

Body weight, feed consumption, serum chemistry, hematology, organ weight and sperm count data were evaluated using the following statistical tests: Bartlett’s test (P<=0.01), one-way analysis of variance (ANOVA) (P<=0.05), and Duncan’s multiple range test (P<=0.05) or Dunnett’s test to indicate statistical significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.
A probability of P<=0.05 (two-tailed) was used to determine significance. If the Bartlett’s test indicated unequal variances, the data were evaluated using the Kruskal–Wallis H-test and the Mann–Whitney U-test.

Details on results:

MORTALITY
No mortality occurred in any of the treated groups during the study.

CLINICAL SIGNS
Animals exposed to 3000 ppm (ca. 14.1 mg/L) had reduced activity levels during exposure that were of generally minor severity. Reduced activity was defined as less movement, decreased alertness, and slower response to tapping on the chamber wall compared with activity levels exhibited by control animals. Signs of diarrhea and red discoloration on the chin hair were also observed. Animals exposed to 1500 ppm (ca. 7.05 mg/L) appeared normal for the first 5 h of day 0 and the first hour or two of days 1 and 2, then exhibited reduced activity of generally minimal severity for the remainder of the daily exposure periods. Reduced activity of minimal severity was generally seen throughout daily exposures thereafter. Control and 500 ppm (2.350 mg/L) animals appeared normal during exposure.

BODY WEIGHT and FOOD CONSUMPTION
Body weights for the 1500 and 3000 ppm groups (ca. 7.05 and 14.1 mg/L) were significantly lower than the control group for most of the study. Overall weight gains for the 3000 ppm group were 62 and 78% of weight gains for the control group (males and females, respectively), while overall weight gains for the 1500 ppm groups were 77 and 70% of the control group (males and females, respectively). Feed consumption for the 3000 ppm groups were significantly lower than for the control group throughout the study for male rats and at all intervals except days 84 and 91 for female rats. Mean feed consumption values for the 1500 ppm groups were significantly lower than the control group for the majority of the study, and sporadically lower for the 500 ppm groups compared with the control group.

CLINICAL CHEMISTRY and HEMATOLOGY
No effects considered as biologically relevant were observed.
No significant differences in hematologic parameters were seen after 30 days on test. Significantly higher mean erythrocyte counts, hemoglobin concentration and hematocrit values were observed for the 3000 ppm male and female rats after 90 days on test compared with the control groups . The mean eosinophil percentage formale 3000 ppm rats was also significantly higher than for the control group. All the values were within normal limits for rats of this age and for the age and strain of the animals used in this laboratory, and none of the differences were considered biologically significant.
After 30 days on test, mean sodium concentrations for the male and female 3000 ppm groups were significantly lower than for the control group. The differences were slight (ca. 1 Meq/L), however. The mean chloride concentration for the 1500 ppm male group was significantly lower compared with the control group. However, this difference was also small (< 4 Meq/L). No other differences in serum chemistries were seen among groups. After 90 days on test, mean albumin and total protein concentrations for the 3000 ppm female group were significantly lower than for the control group. Mean sorbitol dehydrogenase activity for the 1500 ppm male group was significantly higher than for the control group. These changes were not considered to be toxicologically meaningful, and no other differences in serum chemistry were observed among groups.

OPHTHALMOSCOPY
No treatment-related ophthalmologic changes were observed.

ORGAN WEIGHTS
Terminal body weights measured after exsanguination were significantly lower for the 1500 and 3000 ppm male and female groups compared with the control group. Absolute weights of the liver, kidneys and spleen reflect this reduced body weight. Liver and spleen weights for the 1500 and 3000 ppm male and female groups were significantly lower than for the control groups. Absolute kidney weights for the 1500 ppm female and 3000 ppm male and female groups were also significantly lower than for the control groups. However, relative organ weights (to body weight) for these organs were not significantly different with the exception of the spleen-to-body weight ratio for the 3000 ppm male group, which was significantly lower than for the control group.
Reduced body weight was also reflected in the significantly lower absolute brain weight for the 3000 ppm male group and significantly higher brain-to-body weight for the 1500 ppm female and 3000 ppm male groups compared with their respective control groups. In addition, testes-to-body weights for the 1500 and 3000 ppm male groups and the relative lung (to body weight) weight for the 3000 ppm male group were significantly higher than for the control group. Adrenal gland-to-body weight ratios for the 1500 ppm female and 3000 ppm male and female groups were significantly higher than for the respective control groups.

NECROPSY
Exposure-related changes were observed in the nasal passages and stomachs of 1500 and 3000 ppm rats. All male and female 3000 ppm rats and 4/10 male and 6/10 female 1500 ppm rats had necrosis of the olfactory epithelium. Degeneration of the olfactory epithelium was observed along the dorsal medial meatus. Degeneration and regeneration of the epithelium was seen along the third ethmoturbinate and several others. The lesion was characterized by karyorrhexis, pyknosis and depletion of olfactory epithelium cells. The severity of the olfactory lesion was mild to moderate for the 3000 ppm group and minimal to mild for the 1500 ppm group. Olfactory epithelium was replace in some areas by transitional or respiratory epithelium. A few (3/10) 3000 ppm female rats had acute inflammation and degenerative lesions (erosion) of the stomach mucosa (glandular vs forestomach). The severity was minimal to mild.
Lesions of this type in the stomach may be associated with swallowing of mucous containing the test substance, but due to the location of the lesion are more likely caused by stress (Glavin et al., 1991: The neurobiology of stress ulcers. Brain Research Reviews 16, 301–343). An occasional 3000 ppm male rat had atrophy of the thymus, but this was not considered to be a direct compoundrelated effect. Instead, this lesion was attributed to stress (Greaves and Faccini, 1992: Rat Histopathology, A Glossary for Use in Toxicity and Carcinogenicity Studies. p. 51. Elsevier Science, Amsterdam).

SPERM COUNTS
No dose-related or statistically significant effect on epidydimidal or testicular sperm count was observed compared with controls, although the epididymidal sperm counts for all treated groups were lower than controls.

Dose descriptor:

NOAEL

Remarks:

local and systemic

Effect level:

ca. 2.35 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: corresponding to 500 ppm; based on reduced body weight, food consumption and transient CNS effects; signs of necropsy of the olfactory epithelium

Critical effects observed:

not specified

Read-across justifications and data matrices are presented in IUCLID section 13.

Conclusions:

The repeated dose inhalation toxicity study conducted using n-butyl acetate, the metabolic precursor of n-butanol, provides surrogate data for n-butanol. The NOAEL for n-butyl acetate is considered to be 2.35 mg/l. This NOAEL corresponds to 1.52 mg n-butanol /l (corrected for molecular weight).

Executive summary:

The repeated dose inhalation toxicity study conducted using n-butyl acetate, the metabolic precursor of n-butanol, provides surrogate data for n-butanol. Rats (15/sex/dose level) were exposed to vapor concentrations of 0, 2.35, 7.05, 14.1 mg/l n-butyl acetate (i.e. 0, 500, 1500 and 3000 ppm) for 6 hours per workday during 13 weeks. Rats exposed to 7.05 mg/l and 14.1mg/l n-butyl acetate for 13 weeks decreased body weight, decreased weights of certain organ weights, decreased feed consumption were observed. However, no systemic, organ-specific toxicity was observed. Minimal, transient narcosis and sedation effects were also observed in rats exposed to 7.05 mg/l and 14.1 mg/l n-butyl acetate during exposure. No cumulative effect on activity during the 13-week exposure was observed. Thus, the NOAEL for n-butyl acetate is considered to be 2.35 mg/l. The NOAEL of 2.35 mg/l for n-butyl acetate corresponds to 1.52 mg n-butanol /l (corrected for molecular weight).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 520 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The weight of evidence approach is used to determine the hazard caused by repeated administration of tetra-n-butyl titanate, polymer with water. In order to avoid unnecessary animalRelevant data from the substance itself but also from the decomposition products, n-butanol and titanium dioxide (TiO₂) is assessed. Read-across data from the decomposition products is used for assessment, because the target substance is hydrolytically unstable having the half-life less than 2 hours (Brekelmans, M. J. C., 2013). n-Butanol is the most relevant decomposition product of the target substance 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.

Repeated oral toxicity

There is no repeated oral toxicity study conducted for the target substance.Valid experimental data to assess repeated toxicity of n-butanol, the degradation product of the target substance, were available for oral pathway (US EPA, 1986). Four groups of male and female Sprague-Dawley rats (30/sex/group) were administered daily 0, 30, 125 or 500 mg/kg bw/day for either 6 or 13 weeks. CNS effects seen as transient clinical signs (ataxia and hypoactivity) were found at the 500 mg/kg bw/day animals. These effects lasted less than 1 hour and are typical effects of alcohols. The NOAEL in this study was 125 mg/kg/day and the LOAEL was 500 mg/kg/day. No treatment related signs were observed in the 30 mg/kg/day or 125 mg/kg/day treatment groups.

Repeated inhalation toxicity

There are no repeated inhalation studies available for tetra-n-butyl titanate, polymer with water itself. Since the substance hydrolyses rapidly (half-life < 2 hours) when in contact with water or moisture, the intrinsic properties comes from n-butanol, the hazardous degradation product.

There are no robust inhalation toxicity studies available for n-butanol. However, the rapidin vivohydrolysis of n-butyl acetate to n-butanol makes certain n-butyl acetate studies directly applicable to provide surrogate data for n-butanol exposures. The structural analogy of n-butyl acetate (CAS no 123-86-4) and n-butanol (CAS no 71-36-3) has also used to support the human health hazard assessments of these alcohols in OECD SIDS initial assessment reports published by UNEP (2004). Therefore, the toxicity of n-butyl acetate is used in this weight of evidence approach to evaluate the toxicity of the target substance instead of n-butanol data.

The repeated dose inhalation toxicity study conducted using n-butyl acetate provides surrogate data for n-butanol (David et al. 2001). Rats (15/sex/dose level) were exposed to vapor concentrations of 0, 2.35, 7.05 and 14.1 mg/l n-butyl acetate (i. e. 0, 500, 1500 and 3000 ppm) for 6 hours per workday during 13 weeks. Rats exposed to 7.05 mg/l and 14.1 mg/l n-butyl acetate for 13 weeks decreased body weight, decreased weights of certain organ weights, decreased feed consumption were observed. However, no systemic, organ-specific toxicity was observed. Minimal, transient narcosis and sedation effects were also observed in rats exposed to 7.05 mg/l and 14.1 mg/l n-butyl acetate during exposure. No cumulative effect on activity during the 13-week exposure was observed. Thus, the NOAEL for n-butyl acetate is considered to be 2.35 mg/l. The NOAEL of 2.35 mg/l for n-butyl acetate corresponds to 1.52 mg n-butanol /l (corrected for molecular weight).

Based on above information, tetra-n-butyl titanate, polymer with water is regarded not to cause any inhalation hazard.

Repeated dermal toxicity

Dermal route is not considered to be relevant exposure route for humans since 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).

No valid study identified for tetra-n-butyl titanate, polymer with water. The target substance undergoes immediate disintegration (half-life < 2 hours) and after hydrolysis no significant reaction products other than n-butanol and non-hazardous hydrated titanium dioxide (TiO₂) exist.

n-Butanol is regarded not to cause any hazard after dermal application since absorption of the substance through skin is considered negligible (Boman and Maibach, 2000). Furthermore, there is available acute dermal toxicity study conducted for n-butanol showing very low toxicity (5300 mg/kg) (Patty, 1982).

Thus, tetra-n-butyl titanate, polymer with water is regarded not to cause any hazard after repeated dermal application.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
No study available for the substance itself. Based on the read-across data from n-butanol, the most hazardous decomposition product as the target substance is hydrolytically unstable with half-life of < 2 hours (Brekelmans, M. J. C, 2013).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
No study available for the substance itself. Based on the read-across data from n-butyl acetate (metabolic precursor of n-butanol) as the target substance is hydrolytically unstable with half-life of < 2 hours (Brekelmans, M. J. C, 2013).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
No study available for the substance itself. Based on the read-across data from n-butyl acetate (metabolic precursor of n-butanol) as the target substance is hydrolytically unstable with half-life of < 2 hours (Brekelmans, M. J. C, 2013).

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

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

Justification for classification or non-classification

The intrinsic properties of tetra-n-butyl titanate, polymer with water are related to the most hazardous degradation product; n-butanol. Based on the observations made after the subchronic exposures of n-butanol in rats by oral and inhalation routes there is no need for classification of the target substance in accordance with the criteria of CLP Regulation 1272/2008 and the EU directive 67/548/EEC.