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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-04-24 to 1999-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well documented, scientifically sound study that was conducted according to GLP and OECD guideline 406 with no deviations.
Cross-reference
Reason / purpose:
reference to other study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999
Reference Type:
publication
Title:
SIDS Initial Assessment Report for SIAM 21 for Tungsten Carbide (12070-12-1), Washington DC,18-20 October, 2005
Author:
OECD-SIDS
Year:
2005
Bibliographic source:
UNEP Publications
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
1999 guinea pig sensitisation study was available of good quality. Therefore, there is no need to conduct a LLNA study as the guinea pig sensitisation study scientifically fulfills the endpoint. The OECD 406 method provides sensitisation information likely to arise from exposure to test substance via intradermical injection and/or epidermical application to guinea pigs. The guinea pig sensitisation test detects chemicals with moderate to strong sensitisation potential, as well as those with relatively weak sensitisation potential. In such methods activity is measured as a function of challenge-induced dermal hypersensitivity reactions elicited in test animals compared with controls. In addition, guinea pigs have been the animal of choice for predictive sensitisation tests for several decades (way before the LLNA became the test of choice).
The existing guinea pig data submitted here is of good quality as clear results are presented in this robust summary and test methodology followed OECD 406 guidelines, and conducted under GLP. This study it is considered acceptable according to page 266 in ECHA's Chapter r7a on Guidance on Information Requirements and Chemical Safety Assessment.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Tungsten Carbide Powder - Pure
- Physical state: Grey powder
- Analytical purity: >99.98%
- Stability under test conditions: Stable
- Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffs, UK.
- Age at study initiation: 4 to 7 weeks
- Weight at study initiation: 365 to 449g
- Housing: Groups of 5 in suspended metal cages with wire mesh floors
- Diet (e.g. ad libitum): Hay 3 times weekly and Vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) given ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 to 23.5
- Humidity (%): 40 to 59%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 artifical light (0700- 1900 hours) in each 24 hours period.

IN-LIFE DATES: From: March 24, 1998 To: April 24, 1998

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.4 mL of Tungsten Carbide Powder - Pure for the induction topical application and 0.2 mL of Tungsten Carbide Powder- Pure for the topical challenge.
- Concentration (if solution): First induction: intracutaneous injection - 50% w/v
Second induction: occlusive epicutaenous - 75% w/v
Topical challenge: occlusive epicutaneous - 75% % w/v

- The test substance was prepared prior to each applicatiion on the day of dosing.
- The absorption of the test substance was not determined.
- The homogeneity, stability, and purity of the test substance were the responsibility of the Sponsor.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.4 mL of Tungsten Carbide Powder - Pure for the induction topical application and 0.2 mL of Tungsten Carbide Powder- Pure for the topical challenge.
- Concentration (if solution): First induction: intracutaneous injection - 50% w/v
Second induction: occlusive epicutaenous - 75% w/v
Topical challenge: occlusive epicutaneous - 75% % w/v

- The test substance was prepared prior to each applicatiion on the day of dosing.
- The absorption of the test substance was not determined.
- The homogeneity, stability, and purity of the test substance were the responsibility of the Sponsor.
No. of animals per dose:
15 animals total (10 test, 5 control)
Details on study design:
RANGE FINDING TESTS:
- The intradermal and topical irritancy of a range of diluations of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
- Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irritation (Ph. Eur.), approximately one week to the start of the preliminary investigations.

Selection of concentrations of test substance for the main study:
Based on the results of the preliminary investigations, the following concentrations of Tungsten Carbide Powder - Pure were selected:
-Induction intradermal injection: 50% w/v in vehicle; this was the maximum practical concentration for intradermal administration and caused irritation but did not adversely affect the animals.
-Induction topical application: 75% w/v in vehicle.
-Topical challenge: 75 and 37.5% w/v in vehicle.
From preliminary investigation 75% w/v in vehicle was the maximum practical concentration and did not give rise to irritating effects.

MAIN STUDY:
TEST SITE
INDUCTION INTRADERMAL INJECTIONS -Test animals:

A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area .

Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation (Ph.Eur.)
2. Tungsten Carbide Powder - pure, 50% w/v in Alembicol D.
3. Tungsten Carbide Powder - pure, 50% w/v in a 50 : 50 mixture of Freund's complete adjuvant and Alembicol D.

Induction topical application:

Six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by rubbing with0.5 mL per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of Tungsten Carbide Powder-Pure, 75% w/v in Alembicol D. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm " ). This in turn was firmly secured by elastic adhesive bandage ( 50 mm width "Elastoplast" ) wound around the torso of the animal and fixed with an impervious plastic adhesive tape. The dressing was left in place for 48 hrs.

INDUCTION - Control animals:

The control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

OBSERVATIONS:
- Clinical: All animals were observed daily for signs of ill health or toxicity.
- Bodyweight: The bodyweight of each guinea pig on the main study was recorded on Day 1 (day of intradermal injections) and on the last day observations were made of dermal responses to the challenge application.
- Dermal responses: The approximate diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assit in the choice of concentrations for the main study.
Challenge controls:
- The control and test animals were challenged topically two weeks after the topical induction application using Tungsten Carbide Powder- Pure, 75 and 37.5% w/v in Alembicol D.

- Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 mL of Tungsten Carbide Powder - Pure, 75% w/v in Alembicol D and applied to an anterior site on the flank. Tungsten Carbide Powder - Pure, 37.5% w/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hrs under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek" (impervious plastic adhesive tape).

- The challenge sites were evaluated 24 and 48 hours after removal of the patches.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (HCA), benzocaine, and 2-mercaptobenzothiazole

Results and discussion

Positive control results:
Intradermal injections: Slight irritation was seen in test animals at sites receiving HCA, 10% v/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.

Topical application: Slight erythema was observed in test animals following topical application with HCA, as supplied. Slight erythema was seen in the control animals receiving Alembicol D.

Challenge: Dermal reactions seen in all ten test animals were more marked than those seen for controls and therefore all ten test animals were considered to give positive responses.


Conclusion: HCA produced evidence of skin sensitization (delayed contact hypersensitivity) in all of the then animals, thus confirming the sensitivity and reliability of the experimental technique.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.4 mL of Tungsten Carbide Powder-Pure, 75% w/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.4 mL of Tungsten Carbide Powder-Pure, 75% w/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 10.0.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.4 mL of Tungsten Carbide Powder-Pure, 37.5% w/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0.4 mL of Tungsten Carbide Powder-Pure, 37.5% w/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 10.0.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.1 ml of Freund's complete adjuvant 50:50 with Alembicol D
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0.1 ml of Freund's complete adjuvant 50:50 with Alembicol D
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% Hexyl cinnamic aldehyde (HCA) in Alembicol D
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
10% Hexyl cinnamic aldehyde (HCA) in Alembicol D
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis; thickening, dryness.
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Clinical Signs:

No signs of ill health or toxicity were recorded.

Body weight:

Body weight increases were recorded for all guinea-pigs over the period of the study.

Induction:

Intradermal injections:

- Necrosis was recorded at all sites receiving Freund's Complete Adjuvant in test and control animals.

- Slight to well defined irritation was seen in test animals at sites receiving Tungsten Carbide Powder-Pure, 50% w/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.

Topical application:

- Slight erythema was observed in most test animals following topical application of Tungsten Carbide Powder-Pure, 75% w/v in Alembicol D. Black staining was noted over the dosed area, however, it did not interfere with scoring.

- Slight erythema was seen in most of the control guinea-pigs.

Challenge:

- There were no dermal reactions seen in any of the test or control animals, therefore all ten test animals gave negative responses. Black staining was observed on the dose site, however, this did not interfere with scoring.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Tungsten Carbide Powder- Pure did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals.