Lithium potassium titanium oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July-02 August 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study has been performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1992)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

(1996)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(1998)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (MAFF): Japanese Test Guidelines (draft). Test Reports accompanying an application for registration. Unauthorised English translation, July 2000.

Principles of method if other than guideline:

Allergic Contact Dermatitis in the Guinea-Pig: Identification of Contact Allergens" Magnusson B. and Kligman A.M., 1970 published by C.C. Thomas, Springfield, Illinois, USA.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An appropriate guinea pig maximisation test is available which does not justify conducting an additional LLNA due to animal welfare

Test material

Test material information

Constituent 1

Reference substance name:

Lithium Potassium Titanium Oxide

EC Number:

445-620-6

Cas Number:

39318-30-4

Molecular formula:

Hill Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95) CAS Empirical formula: K(0.5-0.7) Li(0.27) Ti(1.73) O(3.8-3.95)

IUPAC Name:

Lithium Potassium Titanium Oxide

Details on test material:

White powder, crystal structure
Expiry date: 31 May 2003
Specific gravity: 3.4
Test substance storage: At room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

- Test animals: Albino guinea pig (SPF-quality). Females, nulliparous and non-pregnant. Ear tattoo.
- Source: Charles River Deutschland, Kisslegg, Germany.
- Age at study initiation: approx. 5 weeks old
- Housing: Group housing of maximally 5 animals per labelled cage (74 cm x 54 cm x 25 cm height) containing purified sawdust as bedding material (SAWI, Jelu Werk, Rosenberg, Germany).
- Diet: Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany).
- Water: Free access to tap water
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions.

Certificates of analysis (sawdust, diet and water) were examined and retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
A controlled environment was maintained in the room with optimal conditions considered as being approximately 15 air changes per hour, a temperature of 21±3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day.

Deviations from the maximum level for temperature (with a maximum of 0.5 ºC) occurred and deviations from the maximum level for relative humidity (with a maximum of 23%) occurred which might have been caused by cleaning procedures in the room. Based on laboratory historical data this protocol deviation was considered not to have affected the study integrity.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

Concentration of test material and vehicle used at induction:
Intradermal injection: 5% test substance in corn oil
Epidermal exposure: 50% test substance in corn oil
Each challenge: 50% test substance in corn oil

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

Concentration of test material and vehicle used at induction:
Intradermal injection: 5% test substance in corn oil
Epidermal exposure: 50% test substance in corn oil
Each challenge: 50% test substance in corn oil

No. of animals per dose:

Experimental group: 10
Control group: 5

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.

Intradermal injections: A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application: A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.

MAIN STUDY:
A. INDUCTION EXPOSURE
Day 1: The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 5% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8: The scapular area between the injection sites was clipped and subsequently treated with 0.5 ml of a 50% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE
Day 21: One flank of all animals was clipped and treated by epidermal application of a 50% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

OBSERVATIONS:
Mortality/viability: twice daily. Toxicity: at least once daily. Body weights prior to start at termination of the study. Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.


Grading Irritation Reactions*:
Erythema and eschar formation:
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Oedema formation:
No oedema 0
Slight oedema (barely perceptible) 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimeter) 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) 4
(*. Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.)

Grading Challenge Reactions:
No visible change 0
Discrete or patchy erythema 1
Moderate and confluent erythema 2
Moderate erythema and swelling 3
Intense erythema and swelling 4


INTERPRETATION
The results for the experimental animals at the challenge phase were compared with the results for the control animals.
All skin reactions will be considered signs of sensitisation provided that such reactions are less severe or are less persistent in the control group.
A sensitisation rate (%) was calculated as follows: the number of sensitised animals as a proportion of the total number of animals in the experimental group.

The results were evaluated according to the EC criteria for classification and labeling of dangerous substances and preparations (Council Directive 67/548/EEC and all adaptions to technical progress and amendments of this Directive published in the Official Journal of the European Communities).


RELIABILITY CHECK
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX . Females of the Dunkin Hartley guinea pig (from Charles River Deutschland, Kisslegg, Germany) were checked for the sensitivity to ALPHA-HEXYLCINNAMICALDEHYDE, TECH. 85%. Intradermal induction: A 20% solution in water (Milli-U, w/w). Epidermal induction: undiluted. Challenge: a 20% solution in water (w/w). The skin reactions in the experimental animals observed in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 50 per cent to the 20% concentration. From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a substance in a Maximisation type of test.

Results and discussion

Positive control results:

A reliability check carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by NOTOX indicated the model functioned properly.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

none

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 %. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.

Results 9

Reading:

other: reliability check carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Remarks:

Model functions properly

Applicant's summary and conclusion

Interpretation of results:

other: Not sensitising

Remarks:

according to EC 1272/2008

Conclusions:

Terracess L does not cause hypersensitivity in the albino Guinea pig by skin contact.
Terracess L does not have to be classified and has no obligatory labelling requirement for sensitisation by skin contact.