Titanium dioxide

Endpoint summary

Description of key information

Titanium dioxide has been tested in two different systems for skin sensitising properties. Both study types show a negative response, thus titanium dioxide does not require classification as skin sensitiser.

Titanium dioxide does not show respiratory sensitising properties in animal studies or in exposure related observations in humans.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-07-18 to 2006-08-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study OECD 429 (2002); no GLP compliance statement.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/JHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Frederick, Maryland, U.S.A.
- Age at study initiation: 10 weeks
- Housing: All animals were housed in stainless steel, wire-mesh cages suspended above cage boards.
- Diet: ad libitum; PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Photoperiod: 12-hour light/dark cycle
No further details are given.

Vehicle:

dimethylformamide

Concentration:

0% (vehicle control), 5%, 25%, 50%, or 100% H-27416 on both ears

No. of animals per dose:

Five groups of 5 mice

Details on study design:

The objective of this study was to evaluate the potential of the test substance to produce a dermal sensitisation response in mice using the local lymph node assay (LLNA).
Twenty-five μL of the test substance were administered topically to the dorsum of each mouse ear for 3 consecutive days (test days 0-2) at dosages listed in the Study Design. One group of mice was similarly dosed with the positive control and one group of mice was similarly dosed with the positive control vehicle. Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-Thymidine per mouse on test day 5.
Approximately 5 hours after the injection, animals were sacrificed by carbon dioxide asphyxiation, draining auricular lymph nodes were removed, and single cell suspensions were prepared. The single cell suspensions were incubated at 2-8°C overnight. On test day 6, the single cell suspensions were counted on a beta counter. The counts per minute (cpm) data were converted to disintegrations per minute (dpm).
A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. The decision process in regard to a positive response includes an SI of greater than or equal to 3.0 together with consideration of dose response and, where appropriate, statistical significance.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Significance was judged at p < 0.05 except for dpm data that were judged at p < 0.01. Lymph node dpm data were transformed to Log to obtain normality or homogenous variances.

Parameter:

SI

Value:

< 3

Test group / Remarks:

25%

Remarks on result:

other: see Remarks

Remarks:

Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. Stimulation indexes (SIs) of less than 3.0 were observed at all test concentrations of the test substance.

Parameter:

SI

Value:

< 3

Test group / Remarks:

50%

Remarks on result:

other: see Remark

Remarks:

Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. Stimulation indexes (SIs) of less than 3.0 were observed at all test concentrations of the test substance.

Parameter:

SI

Value:

< 3

Test group / Remarks:

100%

Remarks on result:

other: see Remark

Remarks:

Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. Stimulation indexes (SIs) of less than 3.0 were observed at all test concentrations of the test substance.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: no data

No statistically significant differences in mean body weights and body weight gains compared to the vehicle control group were observed at any test concentration. No clinical signs of toxicity were observed in the study.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of this study, titanium dioxide (sample: H-27416) did not produce a dermal sensitization response in mice. Based on these data, H-27416 is not a dermal sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-09-06 to 1994-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; self-certified laboratory.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EPA OPP 81-6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Guideline not available/not properly implemented at the date of study conduct.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Ace Animals, Inc., Boyertown, PA 19512, U.S.D.A.
- Weight at study initiation: 355-467 g
- Housing: The animals were housed and maintained in accordance with standards set forth in the Guide for the Care and Use of Laboratory Animals.
- Caging: Stainless steel with elevated wire mesh flooring, 2-3 guinea pigskage.
- Diet: ad libitum; Wayne Guinea Pig Diet
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21.9
- Humidity (%): 51-75
- Photoperiod: 12-hour lighddark cycle
No further details are given.

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Preliminary test
Concentrations: 50 and 100%

Main test
Induction concentration: 100% w/w
Exposure: 6h, occlusive
Challenge concentration: 100% w/w
Exposure: 6h, occlusive

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Preliminary test
Concentrations: 50 and 100%

Main test
Induction concentration: 100% w/w
Exposure: 6h, occlusive
Challenge concentration: 100% w/w
Exposure: 6h, occlusive

No. of animals per dose:

20 guinea pigs

Details on study design:

RANGE FINDING TESTS: Prior to initiation of the study, the irritation potential was determined. 4 unexposed animals were exposed to two concentrations of the test material by the patching technique described in the induction stage. In this test, both sides of the animal were closely clipped and exposed to 0.4 g of the test article moistened with 0.4 ml 0.9% saline (100%) and to 0.2 g of the test article moistened with 0.2 ml of 0.9% saline (50%). The test site was wiped with deionized water following the removal of the patch, examined and scored for irritation according to the Scale for Evaluation of Dermal Irritation (Interpretation of Results section) at 24 and 48 hours from the initial patch application. No dermal irritation was observed at the 100% or the 50% sites. The induction and challenge applications were performed using 0.4 g of the test article (100%) moistened with 0.4 ml 0.9% saline.

MAIN STUDY: Guinea pigs were closely clipped over the induction site on their left flank (approximately 1.5 X 1.5 inches) one day prior to initiation and repeated as necessary.
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Control group: A group of 10 guinea pigs (vehicle control); a 0.4 ml portion of saline was applied. A group of 5 guinea pigs (positive control); a 0.4 ml portion of 0.1% l-chloro-2,4-dinitrobene (DNCB) w/v in 50% ethanol: 0.9% saline was applied.
- Site: onto the prepared skin test site
- Frequency of applications: once a week for three weeks, for a total of three, six-hour applications
- Duration: 6 hours
- Concentrations: 0.4 g moistened with 0.4 ml 0.9% saline
The test site was examined following removal of the patch and scored for irritation according to the Scale for Evaluation of Dermal Irritation at approximately 24 and 48 hours from the initial patch application.

B. CHALLENGE EXPOSURE: After the third induction application, the animals were rested for approximately 14 days. At the termination of the rest period, a challenge application, using the procedures described previously, was applied to a naive challenge site (on the clipped right flank). The challenge
application remained on for approximately 6 hours. Upon removal of the challenge application, the contact area was wiped with deionised water and marked with a color marker to indicate the challenge application site. Dates of application and observations were recorded. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation andor signs of elicited sensitisation.

Challenge controls:

Vehicle control group: After the third induction application, the animals were rested for approximately 14 days. At the termination of the rest period, using the procedures described previously, the vehicle control group was challenged with saline on the clipped left flank. The test article (100%)
was placed on the naive clipped right flank. The challenge application remained on for approximately 6 hours. Upon removal of the challenge application, the contact areas were wiped with deionised water and marked with a color marker to indicate the challenge application sites. Dates of application and observations were recorded. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and/or sips of elicited sensitisation.
Positive control group: After the third induction application, the animals were rested for approximately 14 days. At the termination of the rest period, a challenge application, using the procedures described previously, was applied to a naive challenge site (on the clipped right flank). A group of five naive animals was also treated using procedures described previously at the time of challenge. The challenge application for these groups remained on for approximately 6 hours. Upon removal of the challenge application, the contact area was wiped with deionised water and marked with a color marker to indicate the challenge application site. Dates of application and observations were recorded. Approximately 24 and 48 hours after the challenge application, the sites were examined for dermal irritation and/or signs of elicited sensitisation.

Positive control substance(s):

yes

Remarks:

l-chloro-2,4-dinitrobenzene (99%)

Positive control results:

The positive control (l-chloro-2,4-dinitrobenzene),when compared to the dermal reaction elicited on the naive animals at challenge, is a dermal sensitizer in this study.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100 % test item in vehicle

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100 % test item in vehicle

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100 % test item in vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100 % test item in vehicle

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1% DNCB w/v in 50 %ethanol:0.9 % saline

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

none

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1% DNCB w/v in 50 %ethanol:0.9 % saline

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

none

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

According to the criteria specified by Directive 67/548/EC and subsequent regulations the test item is not considered to be a skin sensitiser and requires no classification.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004-02-06 to 2004-03-18

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Significant methodical deficiencies: - the grading scale included an additional score of 0.5, which is not existent in guideline, therefore deficiencies in data interpretation are adopted and conclusions are questionable

Principles of method if other than guideline:

An 80 % w/w mixture of the test item with mineral oil was topically applied to test guinea pigs, once each week for a three-week induction period. 27 days after the first induction dose, a challenge dose of the test item at its highest non-irritating concentration was applied to a naive site on each guinea pig. A naive control group was treated with the test item at challenge only, Approx. 24 and 48 hr after challenge dose, the animals were scored for erythema.

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

Guideline not available/not properly implemented at the date of study conduct.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elm Hill Breeding Labs, Chelmsford, MA
- Housing: The animals were group housed in suspended stainless steel caging with mesh floors. Litter paper was placed beneath the cages.
- Diet: Pelleted Purina Guinea Pig Chow # 5025
- Water (ad libitum): Filtered tap water

ENVIRONMENTAL CONDITIONS
The animal room was temperature controlled.
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

80% w/w mixture of the test substance

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

80% w/w mixture of the test substance

No. of animals per dose:

Preliminary test: 4 animals
Main test: 20 animals as test animals plus 10 animals as naive animals

Details on study design:

RANGE FINDING TESTS:
A group of animals was used to determine the highest non-irritating concentration (HNIC) of test substance prior to the challenge dose. The fur was removed by clipping (Oster nodel #A5-small) the dorsal area and flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. the test substance was diluted with mineral oil to yield w/w concentrations of 80 %, 75 %, 50 %, and 25 %. Each concentration was applied to a test site using an occlusive 25 mm Hill Top Chamber®, the sites were wrapped with non-allergenic DuraporeTM adhesive tape. After 6 hours of exposure, the chambers were removed and the test sites were gently wiped with ethanol, tap water and clean towel to remove any residual test substance. Prior to the 24 hour evaluation, each guinea pig was closely clipped to facilitate scoring. Approximately 24 hours after application, each site was evaluated for local reactions (erythema) according to the scoring system described below.
From these results, the HNIC (the highest concentration that produced responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero) was established and used for challenge. Based on these results, the HNIC selected for the challenge phase was an 80 % mixture with mineral oil.

MAIN STUDY
- Preparation and selection of animals:
on the day before initiation, the fur of a group of animals removed by clipping the dorsal area and flanks. After clipping and prior to initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy animals without pre-existing skin irritation were selected for test. Animals were reclipped prior to each dose.

- Induction phase:
Once each week for three weeks, four-tenths of a gram of an 80 % w/w mixture of the test substance with mineral oil (to enhance skin contact, the test substance was moistened with mineral oil prior to application) was applied to the left side of each test animal using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic DuraporeTM adhesive tape to avoid dislocation of the chambers and to minimíze loss of the test substance. After the six hour exposure period, the chambers were removed and the test sites were wiped with ethanol, tap water and a clean towel to remove any residual test substance. Approximately 24 and 48 hours after each induction application, each site was evaluated for local reactions (erythema) according to the scoring system as stated below.

- Challenge phase:
Twenty-seven days after the first induction dose, four-tenths of a gram of an 80 % w/w mixture (HNIC) of the test substance with mineral oil (HNIC) was applied to a naive site on the right side of each animal as a challenge dose, using the procedure decribed above. Prior to the 24 hour evaluation, each guinea pig was closely clipped to facilitate scoring. These sites were evaluated for a sensitisation response (erythema) approximately 24 and 48 hours after the challenge application according to the scoring system described below.
In addition to the test animals, 10 guinea pigs from the same shipment were maintained under identical environmental conditions and were treated with the HNIC of the test substance at challenge only.. These animals constituted the "naive" group.

- Scoring system:
0 = no reaction
0.5 = very faint erythema, usually non-confluent
1 = faint erythema, usually confluent
2 = moderate erythema
3 = severe erythema with or without oedema

- Observations:
Body weights: Individual body weights of the animals were recorded prior to initiation and again on the day after challenge application.

- Evaluation:
In order to evalute the sensitization response noted during the challenge phase, two indices were used; one for incidence and one for severity. (Ritz, HArry L. and Buehler, Edwin V. "Planning, Conduct, and Interpretation of Guinea Pig Sensitization PAtch tests, " in Current Concepts in Cutaneous Toxicity, V.A. Drill and P. Lazar (eds.), Academic Press, New York, 1980, P. 25-40).
The incidence index was calculated to evaluate the incidence of erythema (sensitization response) 24 and 48 hours after challenge. The incidence index is used to classify the test substance as a potential contact snesitizer as described below.
The severity index ( sensitization produced) at 24 and 48 hours after challenge was calculated.
The severity index is used as means of summerizing the data and can be applied in situations of questionable negative results to justify rechallenge.
The following criteria were used to classify the test substance as a potential contact sensitizer (Robinson, M., Musair, T., Fletscher, E., and Ritz, H., A review of the Buehler Guinea Pig Skin Sensitization Test and Its Use ina Risk Assessment Process for Human Skin Sensitization in Toxicology, 61, 91 - 107, 1990.)
A. Using the incidence index desscribed above, at the 24 hour and/or 48 hour scoring interval, 15 % or more of the test animals exhibit a positive response (scores > 0.5) in the absence of similar results in the naive control group.
B. The ositive reaction must persist to 48 hours in at least one test animal.

Challenge controls:

10 males were used for the naive group
Dose for challenge application: Four-tenths of a gram of an 80 % w/w mixture of the test substance with mineral oil (highest non-irritating concentration) was applied.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde Technical (HCA)

Positive control results:

Induction phase (HCA technical,applied undiluted,historical positive control animals):
Very faint to faint erythema (0.5 - 1) was noted for eight positive control sites durig the induction phase. Moderate erythema (2) was noted for one site following the third induction.
Challenge phase ( HCA technical, applied as a 75 % w/w mixture in mineral oil, historical positive control animals):
Six of ten positive control animals exhibited signs of a sensitization response, faint erythema (1) 24 hours after challenge. irritation persisted at five sites through 48 hours. very faint erythema (0.5) was noted for all other sites following the challenge dose.
Challenge phase (HCA technical, applied as a 75 % w/w mixture in mineral oil, historical positive naive control animals):
Very faint erythema (0.5) was noted for three of five positive naive control sites 24 hours after challenge. irritation persisted at two sites through 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

Very faint erythema (0.5) was noted at ten of nineteen test sites 24 hours after the challenge dose.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

Irritation persisted at four of these sites through 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Very faint erythema (0.5) was observed at five of ten sites 24 hours after challenge.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Irritation persisted at one of these sites through 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

75 % w/w mixture in mineral oil

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

faint erythema (1) 24 hours after challenge. irritation persisted at five sites through 48 hours. very faint erythema (0.5) was noted for all other sites following the challenge dose.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

75 % w/w mixture in mineral oil

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

faint erythema (1) 24 hours after challenge. irritation persisted at five sites through 48 hours. very faint erythema (0.5) was noted for all other sites following the challenge dose.

One test animal died on Day 12, prior to induction #3. The death of this animal does not appear to be test mateiral related.

Induction phase 8test animals): no dermal irritation noted for any test sites during the induction phase.

Challenge phase: Erythema score (0.5) observed in both the test and naive control animals at challenge were not expected based on the preliminary irritation testing results. Since these are not considered positive responses and incidence and severity were comparable at 24 hours between test and control, these results are considered acceptable and are not indicative of a sensitisation response.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these findings and on the evaluation system used, the test item is not considered to be a contact sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-05-29 to 2003-07-08

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Significant methodical deficiencies: - In deviation to the guideline 10 test and 5 control animals were used - the grading scale included an additional score of 0.5, which is not existent in guideline, therefore deficiencies in data interpretation are adopted and conclusions are questionable

Principles of method if other than guideline:

An 80 % w/w mixture of the test item in mineral oil was topically applied for 6 hr to test guinea pigs, once each week for a 3-week induction period. 28 days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration was applied to a naive site one each guinea pig. A naive control group was treated with the test item at challenge only. Approx. 24 and 48 hours after challenge dose, the animals were scored for erythema.

GLP compliance:

no

Type of study:

Buehler test

Justification for non-LLNA method:

Guideline not available/not properly implemented at the date of study conduct.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elm Hill Breeding Labs, Chelmsford, MA
- Age at study initiation: Young adult
- Weight at study initiation: 387 - 446 g (test group and naive control group)
- Housing: The animals were group housed in suspended stainless steel with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage.
- Diet: Pelleted Purina Guinea Pig Chow # 5025
- Water (ad libitum): Filtered tap water
- Acclimation period: 6 days (Test and naive control animals)

ENVIRONMENTAL CONDITIONS
- Temperature: 19 °C - 22°C
- Relative humidity: 34 %- 58 %
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

80% w/w mixture of the test substance

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

80% w/w mixture of the test substance

No. of animals per dose:

Preliminary irritation group: 4
Test group: 10
Test naive control group: 5

Details on study design:

RANGE FINDING TESTS: The test substance was supplied as 80 %, 75 %, 50 %, and 25 % w/w mixtures in mineral oil.

PREPARATION AND SELECTION OF ANIMALS:
Within 24 hours prior to each application, the fur of each guinea pig assigned to the test group was removed by clipping the dorsal area and flanks. Care was taken to avoid abrading the skin. Prior to study initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy animals without pre-existing skin irritation were selected for test.

INDUCTION PHASE:
Once each week for three weeks, four-tenths of a gram of an 80 % w/w mixture of the test substance in mineral oil (to enhance skin contact, the test substance was moistened with mineral oil prior to application) was applied to the left side of each test animal for six hours using an occlusive 25 mm Hill Top chamber. The chambers were secured in place and wrapped with non-allergenic DuraporeTM adhesive tape to avoid dislocation of the chambers and to minimíze loss of the test substance. After six hour exposure period, the chambers were removed and the test sites were gently cleansed of any residual test substance. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the scoring system as stated below.

CHALLENGE PHASE:
Twenty-eight days after the first induction dose, four-tenths of a gram of an 80 % w/w mixture of the test substance in mineral oil (highest non-irritating concentration) was applied to a naive site on the right side of each animal as a challenge dose, using the methods decribed above. These sites were evaluated for a sensitisation response (erythema) approximately 24 and 48 hours after the challenge application according to the scoring system described below.
In addition to the test animals, five guinea pigs from the same shipment were maintained under identical environmental conditions. These animals were treated with the highest non-irritating concentration of the test substance at challenge only and constituted the "naive control" group.

SCORING SYSTEM:
0 = no erythema
0.5 = very faint erythema, usually non-confluent
1 = faint erythema, usually confluent
2 = moderate erythema
3 = severe erythema with or without oedema

OBSERVATIONS:
Body weights: Individual body weights of the animals were recorded prior to the first induction and again on the day after challenge application.
Clinical observations: The animals were observed daily. if any unusual clinical signs were observed, they were recorded.

EVALUATION:
In order to evaluate the sensitisation response noted during the challenge phase, two indices were used; one for incidence and one for severity. The incidence index was calculated to evaluate the incidence of erythema (sensitisation response) approximately 24 and 48 hours after challenge according to the following:
Scores of 1 or greater in the test group are required to be indicative of sensitisation. If scores of one (1) or greater are seen on the control animals, then the reactions of the test substance group animals that exceed the most severe control reactions are considered to be positive scores.
Incidence is reported as the number of positive animals in each group divided by the total number of animals test in that group.
Severity is reported as the sum of the test grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. All average grades are to be rounded off to the nearest tenth of a unit.

Challenge controls:

5 males were used for the naive control group.
Dose for challenge application: Four-tenths of a gram of an 80 % w/w mixture of the test substance in mineral oil (highest non-irritating concentration) was applied.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde, technical grade (85 % HCA)

Positive control results:

Induction phase (85 % HCA applied undiluted, historical positive control animals):
Very faint to faint erythema (0.5 - 1) was noted for all positive control sites duing the induction phase. Moderate erythema (2) was noted for one site following the third induction.
Challenge phase (85 % HCA applied as a 75 % w/w mixture in mineral oil, historical positive control animals):
Six of ten positive control animals exhibited signs of a sensitisation response (faint erythema (1)) 24 hours after challenge. Similar indications persisted at three sites through 48 hours. Very faint erythema (0.5) was noted for all other sites following the challenge dose.
Challenge phase(85 % HCA applied as a 75 % w/w mixture in mineral oil, historical positive naive control animals):
Very faint erythema (0.5) was noted for two of five positive naive control sites 24 hours after challenge. Irritation cleared from both affected sites by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Very faint erythem (0.5) was noted for five of ten test sites 24 hours after challenge application.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Irritation cleared from all affected sites by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Very faint erythema (0.5) was noted for two of five naive control sites 24 hours after challenge application.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in mineral oil

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Irritation cleared from both sites by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

85 % HCA applied as a 75 % w/w mixture in mineral oil

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

Six of ten positive control animals exhibited signs of a sensitisation response (faint erythema (1)) 24 hours after challenge.Very faint erythema (0.5) was noted for all other sites following the challenge dose.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

85 % HCA applied as a 75 % w/w mixture in mineral oil

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Three of ten positive control animals exhibited signs of a sensitisation response (faint erythema (1)) 48 hours after challenge. Very faint erythema (0.5) was noted for all other sites following the challenge dose.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these findings and on the evaluation system used, the test item is considered not to be a contact sensitiser.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2003-05-27 to 2003-07-11

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Significant methodical deficiencies: - In deviation to the guideline 10 test and 5 control animals were used - the grading scale included an additional score of 0.5, which is not existent in guideline, therefore deficiencies in data interpretation are adopted and conclusions are questionable

Principles of method if other than guideline:

An 80 % w/w mixture of the test item in distilled water was topically applied for 6 hr to test guinea pigs, once each week for a 3-week induction period. 29 days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration was applied to a naive site one each guinea pig. A naive control group was treated with the test item at challenge only. Approx. 24 and 48 hours after challenge dose, the animals were scored for erythema.

GLP compliance:

no

Type of study:

Buehler test

Justification for non-LLNA method:

Guideline not available/not properly implemented at the date of study conduct.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elm Hill Breeding Labs, Chelmsford, MA
- Age at study initiation: Young adult
- Weight at study initiation: 367 - 446 g (test group and test naive control group)
- Housing: The animals were group housed in suspended stainless steel with mesh floors or plastic perforated bottom caging which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals DHEW (NIH). Litter paper was placed beneath the cage.
- Diet: Pelleted Purina Guinea Pig Chow # 5025
- Water (ad libitum): Filtered tap water
- Acclimation period: 4 days (Test and naive control animals)

ENVIRONMENTAL CONDITIONS
- Temperature: 18 °C - 22°C
- Relative humidity: 35 %- 59 %
- Photoperiod (hrs dark / hrs light): 12/12
No further information on the test animals was stated.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

80% w/w mixture of the test substance

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

80% w/w mixture of the test substance

No. of animals per dose:

Preliminary irritation group: 4
Test group: 10
Test naive control group: 5

Details on study design:

RANGE FINDING TESTS: The test substance was supplied as 80 %, 75 %, 50 %, and 25 % w/w mixtures in distilled water.

PREPARATION AND SELECTION OF ANIMALS:
Within 24 hours prior to each application, the fur of each guinea pig assigned to the test group was removed by clipping the dorsal area and flanks. Care was taken to avoid abrading the skin. Prior to study initiation, the animals were weighed and the skin was checked for any abnormalities. Only healthy animals without pre-existing skin irritation were selected for test.

INDUCTION PHASE:
Once each week for three weeks, four-tenths of a gram of an 80 % w/w mixture of the test substance in distilled water (to enhance skin contact, the test substance was moistened with distilled water prior to application) was applied to the left side of each test animal for six hours using an occlusive 25 mm Hill Top chamber. The chambers were secured in place and wrapped with non-allergenic DuraporeTM adhesive tape to avoid dislocation of the chambers and to minimíze loss of the test substance. After six hour exposure period, the chambers were removed and the test sites were gently cleansed of any residual test substance. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the scoring system as stated below.

CHALLENGE PHASE:
Twenty-nine days after the first induction dose, four-tenths of a gram of an 80 % w/w mixture of the test substance in distilled water (highest non-irritating concentration) was applied to a naive site on the right side of each animal as a challenge dose, using the methods decribed above. These sites were evaluated for a sensitisation response (erythema) approximately 24 and 48 hours after the challenge application according to the scoring system described below.
In addition to the test animals, five guinea pigs from the same shipment were maintained under identical environmental conditions. These animals were treated with the highest non-irritating concentration of the test substance at challenge only and constituted the "naive control" group.

SCORING SYSTEM:
0 = no erythema
0.5 = very faint erythema, usually non-confluent
1 = faint erythema, usually confluent
2 = moderate erythema
3 = severe erythema with or without oedema

OBSERVATIONS:
Body weights: Individual body weights of the animals were recorded prior to the first induction and again on the day after challenge application.
Clinical observations: The animals were observed daily. if any unusual clinical signs were observed, they were recorded.

EVALUATION:
In order to evaluate the sensitisation response noted during the challenge phase, two indices were used; one for incidence and one for severity. The incidence index was calculated to evaluate the incidence of erythema (sensitisation response) approximately 24 and 48 hours after challenge according to the following:
Scores of 1 or greater in the test group are required to be indicative of sensitisation. If scores of one (1) or greater are seen on the control animals, then the reactions of the test substance group animals that exceed the most severe control reactions are considered to be positive scores.
Incidence is reported as the number of positive animals in each group divided by the total number of animals test in that group.
Severity is reported as the sum of the test grades divided by the total number of animals tested in a given group determined for both 24 and 48 hours. All average grades are to be rounded off to the nearest tenth of a unit.

Challenge controls:

5 males were used for the naive control group.
Dose for challenge application: Four-tenths of a gram of an 80 % w/w mixture of the test substance in distilled water (highest non-irritating concentration) was applied.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde, technical grade (85 % HCA)

Positive control results:

Induction phase (85 % HCA applied undiluted,historical positive control animals):
Very faint to faint erythema(0.5- 1) was noted for all positive control sites during the induction phase. Moderate erythema 82) was noted for ne site following the third induction.
Challenge phase (85 % HCA applied as a 75 % w/w mixture in mineral oil, historical positive control animals):
Six of ten positive control animals exhibiteed signs of a sensitization response (faint erythema (1)) 24 hours after challenge. Similar indications persisted at three sites through 48 hours. Very faint erythema (0.5) was noted for all other sites folowing the challenge dose.
Challenge phase (85 % HCA applied as a 75 % w/w mixture in mineral oil, historical positive naive control animals):
Very faint erythema (0.5) was noted for two of five positive naive control sites 24 hours after challenge. irritation cleared from both affected sites by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Very faint erythema (0.5) was noted for two of ten sites 24 hours after challenge application.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Test substance applied as an 80 % w/w mixture in distilled water

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Irritation cleared from both sites by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Very faint erythema (0.5) was noted for one of five naive control sites 24 hours after challenge application.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Test substance applied as an 80 % w/w mixture in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Irritation cleared from the affected site by 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

85 % HCA applied as a 75 % w/w mixture in mineral oil

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

Six of ten positive control animals exhibiteed signs of a sensitization response (faint erythema (1)) 24 hours after challenge. Very faint erythema (0.5) was noted for all other sites following the challenge dose.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

85 % HCA applied as a 75 % w/w mixture in mineral oil

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

Three of ten positive control animals exhibiteed signs of a sensitization response (faint erythema (1)) 48 hours after challenge. Very faint erythema (0.5) was noted for all other sites following the challenge dose.

Induction phase (test animals):

Very faint erythema (0.5) was noted for all test sites during the induction phase.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these findings and on the evaluation system used, the test item is considered not to be a contact sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation studies with pigment-grade titanium dioxide

Three studies conducted with pigment-grade titanium dioxide show negative results (Moore, G.E., 2003 a+b and Durando J. 2004). The tests were conducted according to the Bühler method in guinea pigs. All three studies are not considered as reliable due to experimental deficiencies. They deviated from the guideline by using 10 test and 5 control animals only and the grading scale included an additional score of 0.5, which does not exist in guideline. Due to these deficiencies in data interpretation, the conclusions of the studies are regarded as questionable. However, in a weight-of-evidence approach the lack of skin sensitising properties in all three studies on pigment grade titanium dioxide may be considered.

 

Skin sensitisation studies with ultrafine titanium dioxide

Hoban, D. (2006) treated 25 mice with ultrafine titanium dioxide (100% purity: 79% rutile, 21% anatase) in a LLNA test comparable to OECD 429. Mice were divided into 5 groups of 5 animals receiving 0, 5, 25, 50 or 100% test substance to the dorsum of both ears. Statistically significant increases in cell proliferation measurements compared to the vehicle control group were observed at the 50% and 100% test concentrations. Stimulation indexes (SIs) of less than 3.0 were observed at all test concentrations of the test substance.

Moore, G.E. (1994) treated 20 guinea pigs with ultrafine titanium dioxide (95.4% purity) in a modified Bühler test according to OECD 406. Guinea pigs were induced and challenged by occluded application of the test substance to clipped intact guinea pig skin. The same procedures were carried out on a negative control and positive control group. Neither during the induction phase nor 24 and 48 hours after the challenge application of the test substance was redness observed in any of the animals.

Smulders, S. (2015) conducted a mouse local lymph node assay (LLNA) with Titanium dioxide ENPs with an average diameter of 15 nm. Balb/c mice were treated with 0.4, 4 or 40 mg/ml ENP suspension (in 0.9 NaCl) or 4 mg/ml aged paint particles. 2,4 Dinitrochorobenzene was used as positive control. Test days 4-5 were days of rest followed by intravenous injection of 20 μCi of ³H-Thymidine (3HTdR) per mouse on test day 6. Five hours later, the mice were sacrificed by an overdose of Pentobarbital and auricular lymph nodes were removed, pooled and weighed. 3HTdR incorporation was evaluated by β-scintillation counting and was expressed as disintegrations per minute (dpm). The SI for all dose groups was below 2 and thus the test substance did not produce a dermal sensitisation response in mice. Due to some significant methodological deficiencies (e.g. test item insufficiently described, application method, rationale for dose selection) the study is considered as supporting study.

Hussain, S. (2012) conducted a mouse local lymph node assay (LLNA) with Titanium dioxide NPs with an average diameter of 15 nm. Balb/c mice were treated with 0, 0.04 or 0.4 mg/ml NP suspension (in 2.5 mM sodium citrate) or 0.04 mg/ml pigment suspension. 2,4 Dinitrochorobenzene was used as positive control. Test days 3-4 were days of rest followed by intravenous injection of 20 μCi of ³H-Thymidine (3HTdR) per mouse on test day 5. Five hours later, the mice were sacrificed by an overdose of Nembutal (90 mg/kg) and auricular lymph nodes were removed, pooled and weighed. 3HTdR incorporation was evaluated by β-scintillation counting and was expressed as disintegrations per minute (dpm). The SI for all dose groups was below 1.5 and thus the test substance did not produce a dermal sensitisation response in mice. Due to some significant methodological deficiencies (e.g. test item insufficiently described, application method (subcutaneous), rationale for dose selection) the study is considered as supporting study.

Conclusion

Based on the available information it is concluded that titanium dioxide is void of any skin and respiratory sensitising properties:

An LLNA assay and a Buehler test both performed with ultrafine titanium dioxide were negative. In addition, three Buehler tests with pigment-grade titanium dioxide also yielded negative results, despite their shortcomings. By weight-of-evidence, titanium dioxide may therefore be viewed as lacking skin sensitising properties.

Respiratory sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

respiratory sensitisation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

other: not rated acc. to Klimisch

Rationale for reliability incl. deficiencies:

other: Well-documented publication on respiratory sensitisation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Non-allergic (healthy) and ovalbumin-sensitized (asthmatic) female mice were exposed via inhalation to two different sizes of titanium dioxide particles, nanosized and fine , for 2 hours a day, three days a week, for four weeks at a concentration of 10 mg/m^3. Different endpoints were analysed to evaluate the immunological status of the mice.

GLP compliance:

not specified

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS - BALB/c/Sca mice
- Source: Scanbur AB, Sollentuna, Sweden
- Age at study initiation: 7 week old
- Acclimation period: 1 week

Route of induction exposure:

other: intraperitonal

Route of challenge exposure:

inhalation

Vehicle:

other: phosphate-buffered saline (PBS)

Concentration:

10 ± 2 mg/m^3

No. of animals per dose:

8 female mice

Details on study design:

EXPOSURE SYSTEM FOR AEROSOLIZED PARTICLES
The animals were exposed using a solid particle dispenser (Rotating Brush Generator, RBG 1000, Palas, Karlsruhe, Germany) as described by Rossi et al.(2010)*.

AEROSOL AND PARTICLE CHARACTERIZATION
The aerosol size distributions were measured from 10 nm to 1000 nm with a scanning mobility particle sizer (SMPS) consisting of a 63Ni bipolar aerosol neutralizer, a Vienna type differential mobility analyzer (DMA) with length of 28 cm and TSI model 3010 condensation particle counter (CPC) (Wang, 1990)*. Aerosol aerodynamic size distribution was measured from 15.9 nm to 10 μm with an electronic low pressure impactor (ELPI, Dekati) (Marjamäki, 2000). The aerosol mass concentration was determined gravimetrically after collection on nitrocellulose filters (Millipore). Particle concentration inside the particle dispenser chamber was also monitored using a personal DataRAM (online mass concentration meter, pDR1000AN, Waltham, MA, USA).
Aerosolized particles occured as agglomerates. Specific surface area of nTiO2 is 132 m^2/g and 2 m^2/g for fTiO2. The aerodynamic number size distributions indicate that nTiO2 dispersed in air using the solid particle dispenser occurred mostly as agglomerates of 100 nm at a number concentration of 69 000 cm^-3.

MAIN STUDY
- Induction exposure
Mice sensitized intraperitonally with 20 μg of ovalbumin (OVA) in alum (Sigma-Aldrich, St Louis, MO) in 100 μl of phosphate-buffered saline (PBS) on days 1 and 14 of the experiment. Control group was given alum in 100 μl of PBS. Exposure groups were exposed three times a week for 2 hours for the duration of the four week experiment. The exposure concentration was 10 ± 2 mg/m^3 in all tests.

- Challenge exposure
On days 25-27 all mice were challenged with 1% ovalbumin solution via the airways for 20 minutes administered using the ultrasonic nebulizer (DeVilbiss, Glendale Heights, IL).

MEASUREMENTS
A. Measurement of airway responsiveness
Airway responsiveness was measured on day 28 using a single chamber, whole-body plethysmograph system (Buxco, Troy, NY) as described in Hamelmann et al. (1997)*. Mice were exposed to increasing concentrations (1, 3, 10, 30 and 100 mg/ml) of metacholine (Sigma Aldrich) in PBS delivered via an AeroSonic 5000 D ultrasonic nebulizer (DeVilbiss, ITW, Glendale Heights, IL). Before metacholine exposure the baseline is measured for three minutes. After baseline measurements the metacholine is nebulized for 1.5 minutes and airway reactivity is measured for 5 minutes per concentration. Lung reactivity parameters were expressed as Penh (enhanced pause) values. After measurement of lung responsiveness, the mice were sacrificed using an overdose of isoflurane and samples were collected for analysis.

B. Collection of biological samples and preparation
Sample collection and preparation were done as described by Rossi et al. (2010). The following samples were collected: blood serum for antibody analysis, bronchoalveolar lavage (BAL) cells and supernatant for May Grünwald-Giemsa (MGG) staining and protein analysis and lung samples for RNA isolation and hematoxylin and eosin (H&E) and Periodic Acid-Schiff (PAS) staining. The spleens were also dissected from the mice onto 6-well plates with PBS for spleen cell stimulations.

C. Spleen cell stimulations
Spleens were mechanically broken down into paste and filtered to remove larger pieces. Cells were resuspended in RPMI media, counted under light microscopy and plated at a concentration of one million cells/mL. Cells to be stimulated were plated in RPMI containing 100 μg/ml ovalbumin. All cells were incubated for 48 hours after which the supernatant was collected and stored at -70°C prior to analysis.

D. mRNA and protein analyses
- RNA isolation from the lung tissues
The lung samples were homogenized in a FastPrep FP120 (BIO 101, Thermo Savant, Waltham, Mass. USA) -machine and RNA was extracted using the FastRNA Pro Green Kit (Qbiogene/MP Biomedicals, Illkirch, France) and its instructions.
- cDNA synthesis
cDNA was synthesized from 1 μg of total RNA in a 25 ul reaction using MultiScribe Reverse Transcriptase and random primers (The High-Capacity cDNA Archive Kit, Applied Biosystems, Foster City, CA) using the manufacturer’s protocol. The synthesis was performed in a 2720 Thermal Cycler (Applied Biosystems, Carlsbad, California, USA) starting with 25°C for 10 minutes and continuing with 37°C for 120 minutes.
- Polymerase chain reaction (PCR) amplification
PCR primers and probes were ordered as pre-developed assay reagents (18 S rRNA, TNFa, IL-1b, IL-4, IL-13, IL-10, foxp3, CCL3, CXCL5 and CXCL2) from Applied Biosystems (Carlsbad, California). The real-time quantitative PCR was performed as described by Rossi et al. (2010).
- Enzyme-linked immunosorbent assay (ELISA)
TNF-a and IL-13 protein levels from spleen cell supernatants were determined using ELISA kits (eBioscience, San Diego, CA, US) used according to manufacturer’s instructions. An ELISA plate microtiter reader (Multiskan MS, Labsystems, Helsinki, Finland) was used to read the results.
- Luminex
For analysis of TNF-a and IL-13 proteins in BAL fluid supernatants a Milliplex Mouse Cytokine/Chemokine Immunoassay (Millipore Corporation, Billerica, MA) was used according to the manufacturers’ protocol. 3% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO) in PBS was added at a concentration of 0.5% to samples, controls and standards to ensure sufficient protein amounts for the assay. Assay was performed using Luminex xMAP Technology (Bio-Plex 200 System, BioRad, Hercules, CA).

E. Measurement of serum antibodies
For the analysis of OVA-specific IgE, ELISA plates were coated with Purified Rat Anti-mouse IgE antibody (BD Biosciences, San Jose, CA) and incubated overnight. On the second day the plates were blocked with 3% BSA in PBS, serum samples (1:10 and 1:20 dilution) were added to the plate and incubated overnight. Biotinylated OVA was added and streptavidin horseradish peroxidase (Pharmingen 13047E, BD Biosciences) followed by ABTS solution (ABTS Microwell Peroxidase Substrate System, KPL, Gaithersburg, MD). Absorption was read at 405 nm with ELISA plate absorbance reader (Multiskan
MS, Labsystems, Helsinki, Finland).
For OVA-specific IgG2a analysis, ELISA plates were coated with ovalbumin (BD Biosciences) and incubated overnight. On the second day the plates were blocked with 3% BSA in PBS, serum samples (1:60, 1:180, 1:540 and 1:1620 dilution) were added to the plate and incubated overnight. Biotinylated anti-mouse IgG2a (Pharmingen 02012 D, BD Biosciences) was added and streptavidin horseradish peroxidase (BD Biosciences). ABTS solution and peroxidase were mixed 50:50 and added before reading absorption at 405 nm with ELISA plate absorbance reader.

F. Analysis of mucus producing cells
The lung tissue was analysed from formalin-fixed, paraffin-embedded sections. These sections were stained for mucus secreting goblet cells using Periodic acid-Schiff (PAS) -stain. The data was analyzed with Leica Image Manager IM50 version 4.0 (Leica Microsystems Imaging Solutions Ltd., Cambridge, UK). PAS+ cells were counted as an average of PAS+ cells found in 100 μm of bronchus counted from three bronchioles of similar size per mouse (n = 8 mice per group).

G. Hydroxyl radical (•OH) formation capacity
The •OH formation capacity was determined using the benzoic acid probe as described by Rossi et al. (2010).

H. Statistical analysis
The toxicological data was analyzed with the Graph-PadPrism software (GraphPadPrism Software, Inc., San Diego, CA). For all statistical analysis an analysis of variance using one-way ANOVA (nonparametric Kruskal-Walles test) was performed first and when the ANOVA was positive post-testing was performed. The different mice groups were compared using nonparametric Mann-Whitney U - test. P-values less than 0.05 were considered statistically significant.

*Reference
- Rossi EM, Pylkkanen L, Koivisto AJ, Vippola M, Jensen KA, Miettinen M, Sirola K, Nykasenoja H, Karisola P, Stjernvall T, Vanhala E, Kiilunen M, Pasanen P, Mäkinen M, Hämeri K, Joutsensaari J, Tuomi T, Jokiniemi J, Wolff H, Savolainen K, Matikainen S, Alenius H: Airway Exposure to Silica-Coated TiO2 Nanoparticles Induces Pulmonary Neutrophilia in Mice. Toxicol Sci 2010, 113:422-433.
- Wang SC, Flagan RC: Scanning Electrical Mobility Spectrometer. Aerosol Science and Technology 1990, 13:230.
- Marjamäki M, Keskinen J, Chen D-R, Pui DYH: Performance evaluation of the Electrical Low-Pressure Impactor (ELPI). Journal of Aerosol Science 2000, 31:249-261.
- Hamelmann E, Schwarze J, Tadeka K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW: Noninvasive Measurement of Airway Responsiveness in Allergic Mice Using Barometric Plethysmography. Am J Respir Crit Care Med 1997, 156:766-767.

Challenge controls:

Please refer to the field "Details on study design" above

Positive control substance(s):

not specified

Negative control substance(s):

not specified

Results:

- asthmatic mice demonstrate increased numbers of eosinophils and lymphocytes in the airways compared to healthy controls.
- in healthy mice (PBS) there was a significant 4.6-fold increase in the influx of neutrophils following exposure to nTiO2 whereas the numbers of macrophages, eosinophils and lymphocytes remained unaffected.
- numbers of eosinophils and lymphocytes were dramatically reduced in asthmatic mice (OVA) after exposure to nTiO2. Macrophages were reduced in half following nTiO2 and increased 1.7-fold following fTiO2 exposure in asthmatic mice. Changes in numbers of pulmonary neutrophils remained non-significant.
- healthy mice showed clear lungs with no mucus secreting cells whereas PAS+ cells were abundant in the bronchial epithelium of allergic mice.
- TiO2 nanoparticles caused a drastic reduction in the numbers of PAS+ cells in the allergic mice.
- the exposed group showed a statistically significant decrease in goblet cell numbers in the epithelium after the particle exposure.
- exposure of OVA sensitized and challenged mice to nTiO2 reduced airway hyperreactivity (AHR) to the level of healthy mice.
- PBS groups were exposed to TiO2 particles but no difference on airway reactivity to inhaled methacholine was found between them and unexposed PBS groups.
- mRNA expression of proinflammatory cytokines, IL-1b and TNF-a, was downregulated after particle exposure to about half and third respectively in asthmatic mice when compared to the non-exposed control. Th2 type cytokines IL-4 and IL-13, which are present in asthmatic but not in healthy mice, were significantly diminished after exposure to nTiO2.
- nTiO2 exposure reduced IL-10 levels by over 8-fold and Foxp3 levels by 2.3-fold.
- reduction in the protein levels of both TNF-a and IL-13 in the BAL from asthmatic mice exposed to TiO2 particles was observed with statistical significance (one-way ANOVA) in IL-13 levels.
- mRNA expression of proinflammatory CCL3 and neutrophil attracting CXCL5 and CXCL2 was decreased in asthmatic mice after exposure to nanoparticles.
- in healthy mice exposure to nTiO2 caused an almost 5-fold elevation of CXCL5 levels, corresponding to high levels of neutrophils seen in the BALF of the same mice.
- spleen cells from asthmatic nTiO2 exposed mice were either stimulated with OVA or left untreated. OVA stimulated cells express TNF-a and IL-13, but when the mice had been exposed to nTiO2 the protein levels were reduced amply.
- no significant changes in the levels of OVA specific IgE could be seen after exposure to nTiO2 particles.
- levels of OVA specific IgG2a were low and unaffected by particle exposure.

Positive control results:

no data

Negative control results:

no data

Interpretation of results:

not sensitising

Conclusions:

According to the authors, healthy mice elicited pulmonary neutrophilia accompanied by increased chemokine CXCL5 expression when exposed to nTiO2. Allergic pulmonary inflammation was dramatically suppressed in asthmatic mice which were exposed to nTiO2 particles - i.e. the levels of leucocytes, cytokines, chemokines and antibodies characteristic to allergic asthma were substantially decreased.
The authors mentioned that the results suggest that repeated airway exposure to TiO2 particles modulates the airway inflammation depending on the immunological status of the exposed mice.

Reference 2

Endpoint:

respiratory sensitisation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

other: not rated acc. to Klimisch

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

organism used, which are not relevant for exposure; study design (OVA + TiO2) is not relevant for hazard assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study examines the immunological responses in naive male rats and in male rats with ova-induced allergic airway disease following inhalation to titanium dioxide nanoparticles (test item concentration: 10 mg/mL). The responses of two different inbred rat strains were compared: the Dark Aguoti, susceptible to chronic inflammatory disorders, and the Brown Norwegian, susceptible to atopic allergic inflammation.
Naive rats were exposed to an aerosol of the test item for 2 hours/day for 10 days. Another subset of rats was sensitized to the allergen ovalbumin in order to induce airway inflammation. These sensitized rats were exposed to titanium dioxide nanoparticles before and during the allergen challenge. The following parameters were investigated in the rats: respiratory physiology, antibodies in serum, cell differentiation in BALF, as well as cytokine and chemokine levels in BALF and serum.

GLP compliance:

not specified

Specific details on test material used for the study:

not applicable

Species:

rat

Strain:

other: Dark Agouti & Brown Norway

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories (Netherlands)
- Age: 8 - 10 weeks old (Dark Agouti (DA/OlaHsd)); 6 - 8 weeks old (Brown Norway (SsNOlaHsd))
- Housing: housed in groups of four in plastic cages
- Diet (ad libitum): R 36 (Lantmännen Lantbruk, Sweden)
- Water (ad libitum)
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C
- Humidity: 50 - 60 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of induction exposure:

inhalation

Route of challenge exposure:

inhalation

Vehicle:

other: phosphate-buffered saline (PBS)

Concentration:

10 mg/mL

No. of animals per dose:

6 - 8 rats/strain

Details on study design:

EXPERIMENTAL DESIGN
This work was divided in two parts as follows:
1) rats were sacrificed and the samples were analysed one day after the last TiO2 nanoparticles exposure. Respiratory physiology was performed and general health status, antibodies detected in serum, cells differentiation in BALF, measured cytokine, and chemokine levels in BALF were examined.
2) additional rats were included, which were sacrificed and analysed 70 days after the last TiO2 nanoparticles exposure. General health status, antibodies detected in serum, and cell differentiation in BALF were only examined.

In both studies naive rats were exposed to either vehicle control (phosphate-buffered saline (PBS) group) or to TiO2 nanoparticles in a PBS solution (TiO2 group) for 2 h/day for 10 subsequent days.
Additional groups were sensitized with ovalbumin (OVA) on two occasions during the first two weeks and were exposed to three OVA challenges (OVA group) during the last week of the schedule. One group from each strain was exposed to TiO2 nanoparticles for 2 h/day for 10 subsequent days, both before and during the OVA challenges (TiO2/OVA group). The rats were sensitized with two intraperitoneal injections of 100 µg ovalbumin emulsified in sterile saline and Al(OH)3(Alu-Gel-S suspension 1.3%) (1 :3) on days -23 and -10. The sensitized rats were then challenged with aerosolized 1% OVA in water, nose-only for 30 min on days 6, 8 and 10. The TiO2 nanoparticles exposures were performed daily before and during the OVA challenges (days 1-10).

PARTICLE AEROSOL EXPOSURE:
Rats were placed in individual nose-only containers, which were coupled to a Battelle tower connected to a 6-jet Collison nebulizer (flow rate: 7.8 ± 0.2 L/min). The characteristics of the inhaled aerosol, the droplet size distribution and the mass median aerodynamic diameter, were measured at the exit port of the Batelle tower by Malvern Mastersizer X. The agglomerated particle hydrodynamic size distribution within the droplet was measured by collecting the aerosol (flow 1 L/min) in an impinger system and subsequently measuring it with photon cross correlation spectroscopy.
The total deposition of TiO2 nanoparticles in the lungs of the rats was measured after one single exposure in a group of rats (5 rats/strain). The group was dedicated to determination of TiO2 nanoparticles deposition only. To determine the TiO2 content, the lung was digested and diluted before being analyzed with ICP-SFMS for quantification of titanium.
To estimate the deposition fraction in different regions of the respiratory system following inhalation to TiO2 nanoparticles, a multiple path particle dosimetry model was used (MPPD v 2.11, 2013). The deposition fractions of the head, trachea-bronchial, and pulmonary regions were calculated with default settings for the rat model and additional selected settings; a size range of 0.01 µm-10.0 µm, a leaning-forward body orientation with "constant exposure", and "deposition only". In order to calculate deposited mass (% of total aerosol) in different regions of the respiratory system, the deposition fractions derived with MPPD were multiplied with the aerosol size distribution and finally the result was summed over the particle size.

EXAMINATION:
- antibodies in serum: rats were sacrificed and blood samples were collected. Total IgE, OVA specific IgE, and OVA specific IgG were analysed using enzyme-linked immunosorbent assay (ELISA), performed with some modification, according to a previously described protocol (Svensson et al., 2003)*.
- isolation of BALF cells: lungs were lavaged with ice cold Ca2+/Mg2+ -free Hanks balanced salts solution. Two aliquots were collected (2 mL and 23 mL aliquots). After centrifugation, the supernatant from the 2 mL aliquot was used for detection of cytokines and chemokines. The cell pellets from both aliquots were pooled for total cell count using trypan blue exclusion in a Bürker chamber. Differential cell counts of lymphocytes, eosinophils, macrophages, and neutrophils were determined, in duplicate, with cytospin preparations of 30,000 cells stained with May-Grünwald-Giemsa solutions. Three hundred cells were counted from each slide.
- flow cytometry analysis of BALF cells: lymphocytes in BALF were analysed with flow cytometry. B cells were defined as CD45RA+ and CD3--. TH cells as CD4+and CD3+, Tc cells as CD8+' and CD3+, NK cells as NKR-P1A+ and CD3 and NKT cells as NKR-P1A+ and CD3+. Results are presented as total numbers of the different lymphocytes.
- inflammatory cytokines and chemokines in BALF and serum: inflammatory mediators in the BALF and the serum were analyzed. The presence of the following mediators were measured: interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7. IL-10, IL-12(p70), IL-13, IL-17, IL-18, EPO, CSF1, CSF2, CSF3, GRO, IFN-γ, CCL3. CCL20, CCL5, TNF-α and VEGF.
- Measurement of respiratory physiology: twenty four hours after the last exposure, respiratory resistance and compliance were measured following exposure to methacholine (MCh, acetyl-[3-methylcholine chloride). The rats were weighed, anesthetized, tracheostomized, and mechanically ventilated with a small animal ventilator. In order to establish stable baseline respiratory mechanics and to ensure a similar volume history before the experiments, four sigh manoeuvres at 3 x tidal volume were performed at the beginning of the experiment. The rats were then allowed a 2 min resting period before the experiment began. Dynamic respiratory mechanics were measured by applying sinusoidal standardized breathing, and the resulting data were analysed using the single compartment model and multiple linear regression, to give respiratory resistance and compliance. Evaluations of the respiratory mechanics of the peripheral components were made using forced oscillation technique. The primary parameters obtained are tissue resistance and tissue elastance. All parameters were measured continuously and the maximum response to a given concentration of 5-45 mg/mL MCh was determined.

*Reference:
- Svensson, L., Lilliehook, B .. Larsson, R., Bucht.A . 2003. gammadelta Tcells contribute to the systemic immunoglobulin E response and local B-cell reactivity in allergic eosinophilic airway inflammation. Immunology 108, 98-108.

Challenge controls:

Please refer to the field "Details on study design" above.

Positive control substance(s):

not specified

Negative control substance(s):

not specified

Results:

PARTICLE CHARACTERIZATION, EXPOSURE AND DEPOSITION:
1) Particle characterization of titanium dioxide nanoparticles:
- droplet size at the Battelle port: bimodal distribution ranging from 0.5 µm (the cut-off for the instrument) to 10 µm
- Mass median aerodynamic diameter at the Battelle port: 2.12 µm.
- volumetric mean diameter of titanium dioxide nanoparticles within the droplet: about 85 % ranged from 1 to 2.5 µm (1.52 ± 0.16 µm (mean± SD); 15 % ranged from 100 to 200 nm, with an average size close to 100 nm.
2) Deposition:
The deposition of nanoparticles in the lung after one single 2 hour nanoparticles exposure was as follows:
- 168± 12 µg in the Dark Aguoti
- 159 ±48 µg in the Brown Norwegian
Nanoparticles were deposited as follows (decreasing order): pulmonary alveolar region, head (nasal cavity), and trachea bronchiolar region.

No contamination of endotoxin in the titanium dioxide nanoparticles samples were observed.

EXPOSURE IN RATS WITH ALLERGIC AIRWAY INFLAMMATION (sensitive rats)
- general health: no visible signs of impaired general health conditions or reduced body weight in rats after test item exposure in either of the strains when examined on day 11 and day 81.

- antibodies in serum: in both strains, similar levels of allergen-specific lgG were detected in serum on day 11. In the Brown Norwegian rat, ovalbumin induced an increase of ovalbumin-specific-IgE antibodies, which was not observed in Dark Aguoti rats. The total IgE levels in serum did not change after sensitization with ovalbumin, but naive Brown Norwegian rats had higher baseline levels of IgE than naive Dark Aguoti rats. Titanium dioxide exposure of ovalbumin sensitized rats did not result in any significant change of the total lgE, the ovalbumin specific-IgE, or the ovalbumin specific-lgG levels in any of the strains.

- inflammatory responses:
- Dark Agouti rats & Brown Norway rats: OVA-treated rats had increased levels of eosinophils, neutrophils, lymphocytes, and macrophages in the
BALF when compared to naive rats on day 11. The TiO2/OVA-groups showed a decrease in the number of eosinophils compared to the OVA groups. The lymphocytes in the TiO2/OVA-groups were predominately TH cells (CD3+, CD4+) carrying the αβ-TCR receptor. The TiO2/OVA-groups did not affect the numbers of T cells, B cells, or NK cells. The levels of IL-17, CCL20, and CXCL-1 were increased in the BALF of both the OVA- and TiO2/OVA-groups.
- Dark Agouti rats: numbers of neutrophils and lymphocytes had increased following the TiO2 nanoparticles exposure.
- Brown Norway rats: on day 81, 70 days after the last OVA exposure, the number of eosinophils remained elevated. The number of neutrophils was increased in the TiO2/OVA-group compared to the OVA-group (p < 0.01 ). A similar difference was observed in Dark Agouti rats (not statistical significance).

- Measurement of respiratory physiology:
- methacholine (MCh) (15-45 mg/mL): all groups in both strains were hyperreactive to a similar degree.
- methacholine (MCh) (5 mg/mL): differences in AHR between groups and strains were detected.
- Dark Agouti rats: OVA sensitization and the aerosol challenge with OVA resulted in AHR, as indicated by an increase of respiratory resistance, tissue resistance and tissue elastance on day 11 when compared to the control group receiving PBS only. In Brown Norway rats similar differences were observed (not statistical significance). The increase of respiratory resistance in Dark Agouti rats was accompanied by a decrease in respiratory compliance. The TiO2 exposures to the TiO2/OVA-group further decreased respiratory compliance in Dark Agouti rats, compared to the OVA-group.
- Brown Norway rats: no changes in respiratory parameters following TiO2 exposure.

TiO2 NANOPARTICLES EXPOSURE IN NAIVE RATS:
- general health: no visible signs of impaired general health condition or reduced body weight in either of the strains, neither on day 11 nor on day 81.

- inflammatory responses:
- Dark Agouti rats & Brown Norway rats: an increase of neutrophils and lymphocytes in BALF (day 11) was observed, when compared to the control group exposed to PBS only. No significant increase of eosinophils to BALF following TiO2 nanoparticles exposure (day 11) was observed. The expanded Iymphocytes were predominately T helper cells (TH) carrying the αβ T cell receptor.
The day after the last TiO2 nanoparticles exposure, increased concentrations of IL-1α, IL-1β, CXCL1, CSF-1, CCL3, and TNF-α, in the BALF compared to the control group was observed.
On day 81, the number of neutrophils was still elevated in the BALF. Flow cytometry analyses showed elevated numbers of lymphocytes in the TiO2 groups compared to the PBS groups (DA, p < 0.05 and BN p < 0.05). The late-phase lymphocyte response was dominated by TH cells carrying the αβ-TCR receptor. An increase of T cells, carrying the γδ-TCR receptor, and NK cells was also observed.
- Dark Agouti rats: an increase of macrophages in the BALF. Rats displayed increase of IL-6, IL-7, lL-12(p70), IL-13, IL-18, CSF3, CSF2, IFN-γ, CCL20, and VEGF.
- Brown Norway rats: increased levels of Tc cells and NKT cells were observed.

- Measurement of respiratory physiology:
- methacholine (MCh) (5 mg/mL): inhalation of TiO2 nanoparticles in naive Dark Agouti rats resulted in increased respiratory resistance, tissue resistance, and decreased respiratory compliance when compared to rats exposed to PBS only. In Brown Norway rats, TiO2 NPs exposure did not change the respiratory parameters.

Positive control results:

not specified

Negative control results:

not specified

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the authors, naive rats exposed to titanium dioxide nanoparticles developed an increase of neutrophils and lymphocytes in Dark Aguoti rats and Brown Norwegian rats. Furthermore, they stated that airway hyperreactivity and production of inflammatory mediators typical of a T helper 1 type immune response were significantly increased, only in Dark Aguoti rats.
Additionally, the authors mentioned that sensitization of the rats induced a prominent OVA-specific-lgE and lgG response in the Brown Norwegian rats while Dark Aguoti rats only showed an increased IgG response. Also, they stated that sensitized rats of both strains developed airway eosinophilia following allergen challenge, which declined upon exposure to titanium dioxide nanoparticles. The level of neutrophils and lymphocytes increased upon exposure to titanium dioxide nanoparticles in the airways of Dark Aguoti rats but remained unchanged in the airways of Brown Norwegian rats.

The reference is not relevant for human risk assessment. The organism and study design used (OVA + TiO2) are not relevant for hazard assessment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Respiratory sensitisation - Human data

Two epidemiological studies investigated the causative link between titanium dioxide exposure and the onset of lung function impairment/occurrence of chronic respiratory disease.

In a retrospective occupational cohort study by Chen, J.L. & Fayerweather, W.E. (1987 and 1988) a total of male 1576 employees exposed to titanium dioxide were observed from 1956 through 1985 for cancer and chronic respiratory disease incidence. Nested case-control analyses found no statistically significant associations between titanium dioxide exposure and risk of chronic respiratory disease (titanium dioxide OR = 0.8) and chest x-ray abnormalities (titanium dioxide OR = 1.4). No cases of pulmonary fibrosis were found in the titanium dioxide-exposed employees.

In a retrospective occupational cohort study by Ellis et al. (2013, 2010) a total of 3607 male workers employees in three titanium dioxide production facilities were followed form 1935 through 2006. There was no indication of a positive association between occupational exposure to titanium dioxide and non-malignant respiratory disease.

 

Respiratory sensitisation - Animal studies

Rossi et al. investigated the immunological status of non-allergic (healthy) and ovalbumin-sensitized (asthmatic) female mice following inhalation exposure to two different sizes of titanium dioxide particles, nano-sized (silica coated) and fine, for 2 hours a day, three days a week, for four weeks at a concentration of 10 mg/m³. Healthy mice elicited pulmonary neutrophilia accompanied by increased chemokine CXCL5 expression when exposed to ultrafine titanium dioxide. No such response was observed in animals exposed to pigment sized titanium dioxide. Allergic pulmonary inflammation was dramatically suppressed in asthmatic mice, which were exposed to ultrafine titanium dioxide or pigment-grade titanium dioxide particles - i.e. the levels of lymphocytes, cytokines, chemokines and antibodies characteristic to allergic asthma were substantially decreased. The results suggest that repeated airway exposure to titanium dioxide particles does not induce respiratory sensitisation and airway inflammation is modulated depending on the immunological status of the exposed mice.

The publication by Fedulov et al. on respiratory sensitisation exhibits significant reporting and methodological deficiencies. Pregnant and non-pregnant animals received a single intranasal instillation of ultrafine titanium dioxide on PD14. On day 12 -14 post partum, the F1 animals were sensitised by OVA, following pulmonary function analysis. The offspring of pregnant mice treated with titanium dioxide particles showed increased susceptibility to allergy, manifesting as increased airway hyperresponsiveness and allergic inflammation. The P generation was only exposed once and the F1 generation was not exposed towards titanium dioxide. The authors did not confirm an immune response towards titanium dioxide, neither in P nor in F1 generation. It was not shown that the increased susceptibility of allergy was a direct consequence of titanium dioxide sensitisation, since the offspring never seemed to have inhaled titanium dioxide. As a consequence, it is considered doubtful whether any sensitisation to titanium dioxide was actually verified in these animals.

Gustafsson, A. et al. (2014): This study examines the immunological responses in naive male rats and in male rats with ova-induced allergic airway disease following inhalation to ultrafine titanium dioxide (test item concentration: 10 mg/mL). The responses of two different inbred rat strains were compared: the Dark Aguoti, susceptible to chronic inflammatory disorders, and the Brown Norwegian, susceptible to atopic allergic inflammation. Naive rats were exposed to an aerosol of the test item for 2 hours/day for 10 days. Another subset of rats was sensitized to the allergen ovalbumin in order to induce airway inflammation. These sensitized rats were exposed to ultrafine titanium dioxide before and during the allergen challenge. According to the authors, naive rats exposed to ultrafine titanium dioxide developed an increase of neutrophils and lymphocytes in Dark Aguoti rats and Brown Norwegian rats. Furthermore, they stated that airway hyperreactivity and production of inflammatory mediators typical of a T helper 1 type immune response were significantly increased, only in Dark Aguoti rats. Additionally, the authors mentioned that sensitization of the rats induced a prominent OVA-specific-lgE and lgG response in the Brown Norwegian rats while Dark Aguoti rats only showed an increased IgG response. Also, they stated that sensitized rats of both strains developed airway eosinophilia following allergen challenge, which declined upon exposure to ultrafine titanium dioxide. The level of neutrophils and lymphocytes increased upon exposure to ultrafine titanium dioxide in the airways of Dark Aguoti rats but remained unchanged in the airways of Brown Norwegian rats. The reference is not relevant for human risk assessment. The organism and study design used (OVA + titanium dioxide) are not relevant for hazard assessment.

 

Conclusion

Based on the available information it is concluded that titanium dioxide is void of any skin and respiratory sensitising properties:

Concerning some reported indications of a possible respiratory sensitising effect, the findings of Rossi et al. (2010), indicated that titanium dioxide exposure resulted in a modulation of the immunological responses within the lungs of ovalbumin sensitised mice. However, it was not shown whether the exposure resulted in lung deposition of particles in mice. The nanosized (silica-coated) particles displayed a more exacerbated modulation in comparison to the pigment-grade particles. This immunomodulatory effect was much less pronounced in healthy mice. This immunomodulatory effect was mainly presented as a shift of balance from the TH2 profile typically for asthmatic immune responses. However, due to the study design it was not possible to conclude, whether the exposure to titanium dioxide resulted instead in an increased TH1 profile. In general, it has been shown that the exposure to titanium dioxide to sensitised mice resulted in the shift from a humoral to a more innate immune response. This indicates that titanium dioxide does not trigger or enhance respiratory sensitisation in healthy or sensitised mice.

The experimental results published by Fedulov et al. (2008) indicated an increased allergic susceptibility in offspring of mice receiving a single intranasal instillation. However, this causal relationship cannot be concluded based on the study design. It is not clear whether the observed effects of increased airway hyperresponsiveness and increased airway inflammation are a cause of the particle exposure in the P generation or the ovalbumin sensitisation in the F1 generation, since no controls in F1 for effects of the ovalbumin sensitisation protocol were shown. Additionally, it was not shown whether intranasal instillation resulted in systemic availability of particles in the P generation, thus enabling the determination of systemic exposure of the foetus as well. Epigenetic effects, favouring the inherit of allergic susceptibility after titanium dioxide exposure were not evaluated.

 

In accordance with the experimental findings of Gustafsson et al. (2014), titanium dioxide exposure resulted in a slight modulation of immunological responses within the lungs of ovalbumin sensitised rats of two strains. One day after ovalbumin challenge and simultaneous titanium dioxide exposure, a shift in immune cells in BALF compared to non-sensitised, non-exposed and ovalbumin-sensitised non-exposed rats was observed. This shift was mainly characterised by a reduction of immune cells from the adaptive immune response to the more innate immune response (i.e. increase of neutrophils in Dark Agouti rats or decrease of eosinophils in Brown Norway rats). This immunomodulatory effect was mainly presented as a shift of balance from the TH2 profile typically for asthmatic immune responses to a TH1 profile.

Justification for classification or non-classification

Sensitisation

The references Hoban, D.(2006) and Moore, G.E.(1994), are considered as the key studies on skin sensitisation and will be used for classification. The overall sensitisation results are as follows:

Hoban, D.(2006) – Local lymph node assay (LLNA) in mice

Sis of less than 3.0 were observed at all test concentrations of titanium dioxide (5, 25, 50, 100%). Therefore, an EC3 value (the estimated concentration required to induce a threshold positive response, i.e., SI = 3) for the test substance under the conditions of the study was not calculable.

Moore, G.E.(1994) – Buehler method in guinea pigs

The incidence of sensitisation after the challenge application was 0/20. Thus, the classification criteria acc. to regulation (EC) 1272/2008 as skin sensitizer are not met since 0% of the test animals responded.

Both studies were conducted with ultrafine titanium dioxide.

 

Respiratory sensitisation

No bronchial challenge test with titanium dioxide is available. Occupational history data do not indicate a positive association between occupational exposure to titanium dioxide and non-malignant respiratory disease. Thus, there is no evidence from human data that titanium dioxide can lead to specific respiratory hypersensitivity.

None of the repeated dose toxicity studies via inhalation reports respiratory hypersensitivity in the test animals. Repeated airway exposure to nano-sized or fine titanium dioxide particles does not induce respiratory sensitisation in non-allergic (healthy) and ovalbumin-sensitized (asthmatic) female mice. Titanium dioxide did not induce a positive response in the LLNA and thus most probably lacks the potential for respiratory allergy induction.

Based on the above information it is concluded that the classification criteria acc. to regulation (EC) 1272/2008 as respiratory sensitiser are not met for titanium dioxide in the pigment or ultrafine form.