potassium titanium oxide (K2Ti6O13)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 June 1993 to 19 April 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: EPA TSCA Guidelines for Toxicity Studies of Chemicals (1990)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

432-240-0

EC Name:

-

Cas Number:

12056-51-8

Molecular formula:

K2Ti6O13

IUPAC Name:

Potassium hexatitanate

Details on test material:

- Substance type: Solid.
- Physical state in the study: aerosol.
- Lot/batch No.: A-2X16, A-2Z21, A-2822, A-2808, A-3303 and A-3305.
- Expiration date of the lot/batch: No stated expiration date in the lots received.
- Storage condition of test material: At ambient room temperature in the testing laboratory's compound repository.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Raleigh Laboratory, Raleigh, NC, USA.
- Age at study initiation: 6 to 8 weeks old.
- Fasting period before study: No, except during exposure.
- Housing: All animals were housed individually in stainless steel lids, wire-bottom cages.
- Diet: Ad libitum (except during exposure), certified Purina Rodent Chow in pellet form.
- Water: Ad libitum (including during exposure), mains water (City of Columbus, which conforms with the EPA standards).
- Acclimation period: Maximum 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC.
- Humidity (%): Probably 40 - 70%.
- Air changes (per hr): At least 10 changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06:00 to 18:00) and 12 hours darkness each day during the study using fluorescent lighting.

IN-LIFE DATES
- Base study group: From: Day 0 To: Day 92.
- Recovery group: From: Day 0 To: Day 183 (approx).
- Lung clearance group: From: Day 0 To: Day 92/Day 134 (approx)/Day 176 (approx).

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: not applicable

Remarks on MMAD:

MMAD / GSD:

5mg/m3: Mean fibre length = 2.01 µm, mean fibre width = 0.66 µm

50mg/m3: Mean fibre length = 1.91 µm, mean fibre width = 0.65µm

500mg/m3: Mean fibre length = 1.92 µm, mean fibre width = 0.69µm

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazleton H1000 and H2000 stainless steel and glass exposure chambers.

- System of generating particulates/aerosols: Accurate Model 302 Dry Chemical Feeder (Accurate, Inc., Whitewater, WI).

- Temperature (°C): 22.0 - 22.4ºC.

- Humidity (%): 55.8 - 65.4%.

- Air flow rate: 497.7 - 502.8 L/min.

- Method of particle size determination: Test atmosphere fibre count and fibre size distribution were measured in the exposure atmospheres. Fibre concentration (fibres/cm3) was calculated by dividing the number of fibres present in the field or fields of view by the product of the ratio of field size to the total collection area of the filter by the total volume of air sampled through the filter.


TEST ATMOSPHERE
- analytical method used: gravimetric mass concentration analysis.
- Samples taken from: multiple locations within the exposure planes of the chamber.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure system aerosol concentrations were assessed as total mass concentrations by gravimetric filter analysis and total fibre count. Total mass concentrations were measured using Gelman #66075, 25 mm, glass fibre filters (Gelman Sciences, Ann Arbor, MI) placed into open-face filter holders and inserted into exposure ports within the chambers. Immediately after sampling, the filters were weighed and the mass concentration of the total aerosol was calculated from the accumulated mass and the sample volume.
Relative SD of the grand mean aerosol mass concentrations were less than 20% indicating the concentrations were stable over time.

Duration of treatment / exposure:

91 days.

Frequency of treatment:

Duration of exposure per day: 6 hours
Dosing regime: 5 days/week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5 mg/m3
Basis:
other: aerosol in the air, 0.005 mg/l

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/m3
Basis:
other: aerosol in the air, 0.05 mg/l

Doses / concentrations 3

Remarks:

Doses / Concentrations:
500 mg/m3
Basis:
other: aerosol in the air, 0.5 mg/l

No. of animals per sex per dose:

50 animals per sex per dose and the control group:
Male: 50 animals at 0 mg/l
Male: 50 animals at 0.005 mg/l
Male: 50 animals at 0.05 mg/l
Male: 50 animals at 0.5 mg/l
Female: 50 animals at 0 mg/l
Female: 50 animals at 0.005 mg/l
Female: 50 animals at 0.05 mg/l
Female: 50 animals at 0.5 mg/l

Base study group:
10 animals per sex per dose and the control group, recovery group
10 animals per sex per dose and the control group, initial lung clearance group
15 animals per sex per dose and the control group, repeat lung clearance group
15 animals per sex per dose and the control group

Control animals:

other: yes, filtered, conditioned room air.

Details on study design:

The inhalation exposure route was chosen for this sub-chronic toxicity study, from the indications from the acute toxicity studies, physico-chemical properties and likely human exposure.

- Dose selection rationale: Based on the 14-Day repeated dose range finder study. See Section 7.5.3. Repeated dose toxicity: 14-day study.

Post-exposure recovery period in satellite groups:
- Recovery group: From: Day 0 To: Day 182 (approx).
- Lung clearance group: From: Day 0 To: Day 91/Day 133 (approx)/Day 175 (approx).

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes
Twice daily ( at least 6 hours apart) during the study period for mortality, moribundity, and once daily during th exposure and recovery period for clinical signs.

BODY WEIGHT: Yes
Individual bodyweights were recorded on Day 1 and then weekly thereafter, and at scheduled necropsy.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
Blood samples were obtained via vena cava at necropsies. Blood samples were collected in the tube with EDTA (anticoagulant) for haematology, and without EDTA for clinical chemistry.
- Anaesthetic used for blood collection: Propylene glycol-free sodium pentobarbital.
- How many animals: 20 males and 20 females (all base study and recovery group rats).
- Parameters checked in tables 4 -10 which are attached were examined.


URINALYSIS: Yes
- Time schedule for collection of urine: Overnight (12 - 16 hour sample) from the base study rats during Week 12 and from the recovery group rats during Week 25.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No.
- Parameters checked: Total volume, appearance, pH, specific gravity, glucose, creatinine, urea nitrogen, protein, ketones, urobilinogen, occult blood and microscopic examination of urine sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete gross necropsy was performed on all base study rats the day following their final exposure and all recovery group animals at the end of the 13-week recovery period. All rats were weighed prior to necropsy and were humanely killed by exsanguination after administration of pentobarbital anesthesia. Each necropsy included examination of the external surface of the body and an examination of all required tissues, with special attention given to the lungs and upper respiratory tract. The tissues in tables 12 -19 along with any identified gross lesions were preserved in 10% neutral fixative. After the organ weight was determined, lungs were perfused with 10% formalin using a gravity filling device. The trachea was ligated after infusion to ensure trapping of fixative in airways and alveoli.

Organ weights:
The following organs were weighed from all animals surviving tthe scheduled necropsy from the base and recovery groups. Each of the following organs were removed, trimmed of extraneous tissue, and weighed: lungs, liver, kidneys, adrenals (paired), brain and testes/ovaries.
A wet and dry lung weight was collected from all animals from the lung clearance groups.


HISTOPATHOLOGY: Yes
Histopathological examinations were conducted on all tissues from the base study group animals from the high concentration exposure group and the air control group. Examinations were also conducted on tissues from the recovery group animals from the high concentration exposure group and the air control group. The lungs and upper respiratory tract were identified as the target organs and were examined from the middle and low concentration groups. The lungs were sectioned to present a maximal section of the mainstem bronchi. The nose and nasal turbinates were prepared in four sections using the landmarks. In addition to the first review of slides, a blind re-evaluation of the target organ slides was performed.

Other examinations:

LUNG CLEARANCE STUDIES

At the end of the 13-week exposure period, a subgroup of animals was used to measure the total titanium (Ti) concentration within the lung. Ti was used as the marker for fibre lung burdens. Five rats/sex/group from the lung clearance subgroup were necropsied at the intervals;
Within 24 hours of their last exposure of the 91-day exposure period;
Near the end of the 6-week post-exposure recovery; and
Near the end of the 12-week post-exposure recovery period.
The lungs from all rats were dissected and the fresh weights were determined. Then each lung was placed in a labelled megacassette and dried in a forced air oven overnight and the dry lung weights were recorded. All samples were stored at room temperature until the analysis fro Ti.
Each lung was digested in an acid solution and formulated in an appropriate matrix for atomic absorption analysis of Ti. The concentration of Ti per unit mass dry lung and wer lung was calculated along with the total lung burden of Ti.
The lung clearance portion of the study was repeated because some lung tissue was inadvertently discarded before analysis.

Statistics:

Normally parametric data were by variance and pairwise comparisons made between test groups and controls using Dunnett’s t-test. Nonparametric data were analysed by a modified t-test. These analyses were performed using software within the Xybion Data system.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no unscheduled animal deaths in this study. No treatment-related clinical findings were observed.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled animal deaths in this study. No treatment-related clinical findings were observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The weight changes in the 0.5 mg/L group are considered to be a non-specific effect, probably related to the high dose response of inhaled foreign material, rather than to any direct specific toxic effect of the compound.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no haematological effects considered treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no blood chemistry effects considered treatment-related.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant urinary effects observed.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

lncreased absolute lung weight values were observed in. m ales at 0.05 mg/l (16%) and both sexes at 0.5 mg/l (59% in males, 20% in females). After a 13 week recovery period, increased lung weights were observed only in the 0.5 mg/l females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations in the lungs related to fibre exposure were observed only in the high concentration groups. Diffuse discolouration of the lungs, local nodular enlargement and/or discolouration of thymic lymph nodes or areas of the thymuses and bronchiolar lymph nodes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Particle-laden macrophages in the lungs, thickening of the alveolar walls.

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY
There were no unscheduled animal deaths in this study.

CLINICAL OBSERVATIONS
See results in Table 1, attached. No treatment-related clinical findings were observed. Red eye discharge and corneal opacity and alopecia observed in the lung clearance group only which is nonspecific and/or lack dose response therefore considered not to be related to test substance exposure.

BODY WEIGHT See results in tables 2 and 3, attached. Mean bodyweights were up to 8% lower than controls in the male low and high exposure and recovery groups and in the female high concentration group. The slight decreases were sporadic in the low exposure group and not consistent in the high exposure group for either sex. The degree of bodyweight loss did not increase with either the fibre concentration or with duration of exposure. The weight changes in the 0.5 mg/l group are considered to be a non-specific effect, probably related to the high dose response of inhaled foreign material, rather than to any direct specific toxic effect of the compound.

HAEMATOLOGY / CLINICAL CHEMISTRY
See results in tables 4 - 10, attached. There were no haematological effects considered to be treatment-related. There were no blood chemistry effects considered to be treatment-related.

URINALYSIS
See results in Tables 11, attached. There were no toxicologically significant urinary effects observed.

GROSS PATHOLOGY
See results in tables 12 and 13, attached. Macroscopic observations in the lungs related to fibre exposure were observed only in the high concentration groups. Diffuse discolouration of the lungs was seen in all 0.5 mg/l group rats after thirteen weeks of exposure and remained present after thirteen weeks of recovery. In these same rats, a number of thymic lymph nodes or areas of the thymuses appeared to have test substance exposure-related findings consisting of either a focal nodular enlargement and/or discolouration. In addition one 0.05 mg/l female and one male and two females exposed at 0.5 mg/l had bronchiolar lymph nodes that appeared to be enlarged or had a nodule. Subsequent microscopic examination revealed that these lymphoid tissues were laden with particle filled macrophages. All other gross observations were spontaneous, incidental lesions that were not related to exposure.

ORGAN WEIGHTS
See results in tables 14 - 17, attached. Increased absolute lung weight values were observed in males at 0.05 mg/l (16%) and both sexes at 0.5 mg/l (59% in males, 20% in females). After a 13-week recovery period, increased lung weights were observed only in the 0.5 mg/l females.

HISTOPATHOLOGY: NON-NEOPLASTIC
See results in tables 18 and 19, attached. Test substance exposure-related effects at the end of the 13-weeks of treatment were found in the lungs. Distinct concentration dependent histopathology changes consisted of particle-laden macrophages (>= 0.005 mg/l) that were intraluminal, as well as, being present within the interstitium of the alveoli. This latter observation, in association with increased numbers of alveolar epithelial cells and interstitial cells, was the cause for minimal to mild thickening of the alveolar walls at fibre concentrations >= 0.05 mg/l following exposure, which was also present in the recovery group rats. Although there were occasional focci of interstitial cell proliferation, there was no obvious mature collogen deposition (fibrosis) as determined by conventional hematoxylin and eosin staining. The multifocal distribution of thickened alveolar walls within both the exposure and recovery groups appeared to be an indication of slight localised irritation caused by the particulate being phagocytised. Little inflammatory response was present. The severity of the thickening of the alveolar walls was not concentration dependent at either the interim or final necropsies and there was no significant evidence of progression of severity of these lesions over the 3 month recovery period. There was evidence of clearance of particulate material from the lungs in the high concentration groups. During the exposure phase the average severity of the pulmonary particle-laden macrophages for the base rats ranged from minimal (grade 1) in the 0.005 mg/l group to severe (grade 4) in the 0.5 mg/l group. Following recovery, the only difference was in the 0.5 mg/l group rats where the severity for the particle-laden macrophages ranged from moderate (grade 3) to severe. A lesion termed alveolar epithelial hyperplasia and characterised as focal hyperplasia of epithelial cells of the alveoli was present in one 0.005 mg/l group female and one 0.5 mg/l group male at the conclusion of thirteen of exposure. Particle-laden macrophages were present in a few enlarged bronchiolar lymph nodes of 0.5 mg/l exposed rats and in thymic lymph nodes that were found for exposed-rats. The average severity of the particle-laden macrophages in the thymic lymph nodes was exposure-related and similar to findings in the lungs. These findings are compatible with lymphatic clearence of particulate material from the lungs to regional lymph nodes.
Comparison of lung and thymic lymph node findings to those determined during the blind phase of the study revealed very similar results, indicating that prior knowledge of the concentration groups had not biased the study pathologist.

HISTOPATHOLOGY: NEOPLASTIC
A small alveolar/bronchiolar adenoma in the lungs of a 0.05 mg/l group male allowed to recover 13 weeks after the conclusion of exposure was observed. Although possessing some mitotic figures and infiltrating around a blood vessel, this tumour was viewed as exhibiting expansive growth rather than invasiveness. The presence of a single adenoma in one rat from the mid-exposure level group is inconclusive.

LUNG CLEARANCE RESULTS See results in tables 20 and 21, attached. There was a clear, aerosol exposure concentration dependent, increase in total Ti lung content and Ti lung tissue concentration. Rats exposed to 0.005 mg/l had significantly lower Ti lung levels compared to rats exposed to 0.05 mg/l, which had titanium levels approximately 12 times greater than those exposed at 0.005 mg/l. Rats exposed to 0.5 mg/l had titanium levels approximately 5 times greater than those exposed at 0.05 mg/l at the 0-week post-exposure necropsy. Background or endogenous Ti lung levels in control rats were approximately 2 orders of magnitude lower than rats exposed to the lowest aerosol concentration (0.005 mg/l). The greatest degree of clearance of Ti from the lungs occurred in rats at the lowest aerosol concentration.

Effect levels

1

Dose descriptor:

NOAEC

Effect level:

0.5 other: mg/L/6h/day

Based on:

other: aerosol of test material in air

Sex:

male/female

Basis for effect level:

other: Addressed in the NONS dossier.

2

Dose descriptor:

NOAEC

Effect level:

500 mg/m³ air

Based on:

other: aerosol of test material in air

Sex:

male/female

Basis for effect level:

other: Addressed in the NONS dossier.

3

Dose descriptor:

NOAEC

Effect level:

0.005 other: mg/L/6h/day

Based on:

other: Aerosol test material in air

Basis for effect level:

other: Conclusions drawn in the original study report, which is equivalent of 0.005 mg/L/6/h/day. The lack of systemic or respiratory tract toxicity at the concentration.

4

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air

Based on:

other: aerosol of test material in air

Sex:

male/female

Basis for effect level:

other: Conclusions drawn in the original study report, which is equivalent of 0.005 mg/L/6/h/day. The lack of systemic or respiratory tract toxicity at the concentration.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Bodyweight:

Mean bodyweights were up to 8% lower than controls in the male low and high exposure and recovery groups and in the female high concentration group. The slight decreases were sporadic in the low exposure group and not consistent in the high exposure group for either sex. The degree of bodyweight loss did not increase with either the fibre concentration or with duration of exposure.

Lung clearance results:

There was a clear, aerosol exposure concentration dependent, increase in total Ti lung content and Ti lung tissue concentration. Rats exposed to 0.005 mg/1 had significantly lower Ti lung levels compared to rats exposed to 0.05 mg/1, which had titanium levels approximately 12 times greater than those exposed at 0.005 mg/1. Rats exposed to 0.5 mg/1 had titanium levels approximately 5 times greater than those exposed at 0.05 mg/1 at the week.post-exposure necropsy.

Background or endogenous Ti lung levels in control rats were approximately 2 orders of magnitude lower than rats exposed to the lowest aerosol concentration (0.005 mg/1).

Effects in organs: Necropsy:

Macroscopic observations in the lungs related to fibre exposure were observed only in the high concentration groups.

Diffuse discolouration of the lungs, was seen in all 0.5 mg/1 group rats after thirteen weeks of exposure and remained present after thirteen weeks of recovery. In these same rats, a number ofthymic lymph nodes or areas of the thymuses appeared to have test substance exposure-related findings consisting of either a focal nodular enlargement and/or discolouration.

In addition one 0.05 mg/1 female and one male and two females exposed at 0.5 mg/1 had bronchiolar lymph nodes that appeared to be enlarged or had a nodule.

Subsequent microscopic examination revealed that these lymphoid tissues were laden with particle filled macrophages.

All other gross observations were spontaneous.

Histopathology

Test substance exposure-related effects at the end of the 13-weeks of treatment were found in the lungs. Distinct concentration dependent histopathology changes consisted of particle-laden macrophages (>=0.005 mg/1) that were intraluminal, as well as, being present within the interstitium of the alveoli. This latter observation, in association with increased numbers of alveolar epithelial cells and interstitial cells, was the cause for minimal to mild thickening of the alveolar walls at fibre concentrations >= 0.05 mg/1 following exposure, which was also present in the recovery group rats. Although there were occasional focci of interstitial cell proliferation, there was no obvious mature collogen deposition (fibrosis) as determined by conventional hematoxylin and eosin staining.

The multifocal distribution of thickened alveolar walls within both the exposure and recovery groups appeared to be an indication of slight localized irritation caused by the particulate being phagocytized. Little inflammatory response was present. The severity of the thickening of the alveolar walls was not concentration dependent at either the interim or final necropsies and there was no significant evidence of progression of severity of these lesions over the 3 month recovery period.

There was evidence of clearance of particulate material from the lungs in the high concentration groups. During the exposure phase the average severity of the pulmonary particle-laden macrophages for the base rats ranged from minimal (grade 1) in the 0.005 mg/1 group to severe (grade 4) in the 0.5 mg/1 group. Following recovery, the only differente was in the 0.5 mg/1 group rats where the severity for the particle-laden macrophages ranged from moderate (grade 3) to severe.

A lesion termed alveolar epithelial hyperplasia and characterised as focal hyperplasia of epithelial cells of the alveoli was present in one 0.005 mg/1 group female and one 0.5 mg/1 group male at the conclusion of thirteen of exposure.

A small alveolar/bronchiolar adenoma in the lungs of a 0.05 mg/1 group male allowed to recover 13 weeks after the conclusion of exposure was observed. Although possessing some mitotic figures and infiltrating around a blood vessel, this tumor was viewed as exhibiting expansive growth rather than invasiveness. The presence of a single adenoma in one rat from the mid-exposure level group is inconclusive.

Particle-laden macrophages were present in a few enlarged bronchiolar lymph nodes of 0.5 mg/1 exposed rats and in thymic lymph nodes that were found for exposed-rats. The average severity of the particle-laden macrophages in the thymic lymph nodes was exposure-related and similar to findings in the lungs. These findings are compatible with lymphatic clearence of particulate materia! from the lungs to regionallymph nodes.

Comparison of lung and thymic lymph node findings to those determined during the blind phase of the study revealed very similar results, indicating that prior knowledge of the concentration groups had not biased the study pathologist.

Applicant's summary and conclusion

Conclusions:

The NOAEL in this study was considered to be 5 mg/m3 (2300 – 2400 f/cm3) in the original report, based on the lack of systemic or respiratory tract toxicity at this exposure concentration. The NOAEL addressed in the NONS dossier was 0.5 mg/L/6/h/day.
The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).

Executive summary:

The NOAEL in this study was considered to be 5 mg/m³ (2300 – 2400 f/cm³) in the original report, based on the lack of systemic or respiratory tract toxicity at this exposure concentration. The NOAEL addressed in the NONS dossier was 0.5 mg/L/6/h/day.

The test material does not meet the criteria for classification according to EU classification system (Council Directive 67/548/EEC and Regulation (EC) No 1272/2008).