Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9th October to 7th November 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in accordance with recognised guideline. Although no data is available on the study's GLP compliance equivalent quality assurance methods were in place at the testing laboratory. The substance is present in an oil formulation at ca.30%. No positive control used.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
The test group consisted of 20 animals.
GLP compliance:
not specified
Remarks:
Quality assurance audit and study director statement are included in the report
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Dinoseb (DNBP) in oil 300 g/l.
30-31.2% w/w DNBP.
DNBP formulation, a brown liquid, was received on 11 July 1984 and was stored at ambient temperature.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England.
- Weight at study initiation: 360-430g
- Housing: the guinea-pigs were housed in suspended cages with wire mesh floors
- Diet/water (e.g. ad libitum): free access to tap water and a Vitamin C-enriched Guinea-Pig Diet F.D.1 (Special Diets Services Limited).
Hay was given once weekly.
- Acclimation period: the guinea-pigs selected for the study were all acclimated to the laboratory environment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21°C
- Humidity (%): 30-70%
- Air changes (per hr): Air exchange was maintained at approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): lighting was controlled by means of a time switch to give 12 hours of artificial light in each 24 hour period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction:
0.05% v/v in water for injection & 5% v/v in water for topical patch application (48 hrs exposure)

Challenge:
5% v/v in water for patch application (24 hrs exposure)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction:
0.05% v/v in water for injection & 5% v/v in water for topical patch application (48 hrs exposure)

Challenge:
5% v/v in water for patch application (24 hrs exposure)
No. of animals per dose:
20 females for test dose group & 10 females for Freund's control group
Details on study design:
RANGE FINDING TESTS:
The intradermal and topical irritancy of a range of aqueous dilutions of DNBP formulation was investigated to identify
(a) irritant test substance concentrations suitable for the induction phase of the main study and
(b) non-irritant concentrations by the topical route of administration for the challenge phase.

Using these criteria, the following concentrations of DNBP formulation would have been selected:
Induction: Intradermal injection: 0.05% v/v in water for injection.
Topical application: DNBP formulation, as supplied.
Challenge: 10% and 5% v/v in distilled water.

However, when these concentrations were utilised for the main study, all the test animals were found dead the day after the induction topical application of DNBP formulation, as supplied.

The study was therefore repeated using lower concentrations of DNBP formulation to avoid problems of toxicity and allow completion of the test.

The following concentrations of DNBP formulation were selected for the repeat test:
Induction:
Intradermal injection: 0.05% v/v in water for injection.
Topical application: 5% v/v in distilled water.

Challenge: 5% v/v in distilled water.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal injections:
A 4 x 6 cm 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 simultaneously into this area as shown in Figure 1.

Injectables were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for injection.
2. DNBP formulation, 0.05% v/v in water for injection.
3. DNBP formulation, 0.05% v/v in a 50 : 50 mixture of Freund's complete adjuvant and water for injection.

Topical application:
One week after the injections, the same 4 x 6 cm interscapular area was clipped and shaved free of hair.
A 2 x 4 cm patch of Whatman No. 3 paper was saturated with DNBP formulation, 5% v/v in distilled water. The patch was placed on the skin and covered by a length of impermeable plastic adhesive tape (5 cm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage ("Elastoplast" 5 cm width) wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.


B. CHALLENGE EXPOSURE
The test and control animals were challenged topically two weeks after the induction period using DNBP formulation, 5% v/v in distilled water.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 2 x 2 cm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of DNBP formulation, 5% v/v in distilled water and applied on the flank.
The patch was sealed to the flank for 24 hours under a strip of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".


Challenge controls:
During the induction period, the control animals were treated similarly to the test animals, with the exception that the test compound was omitted from the intradermal injections and topical application.
Positive control substance(s):
not specified

Results and discussion

Positive control results:
No data

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5% v/v test material in water
No. with + reactions:
20
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5% v/v test material in water. No with. + reactions: 20.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Freund's treated control
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Freund's treated control. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5% v/v test material in water
No. with + reactions:
20
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% v/v test material in water. No with. + reactions: 20.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Freund's treated control
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Freund's treated control. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
5% v/v test material in water
No. with + reactions:
20
Total no. in group:
20
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5% v/v test material in water. No with. + reactions: 20.0. Total no. in groups: 20.0.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Remarks on result:
other: Reading: other: 3rd reading. . Hours after challenge: 72.0. Group: test group.

Any other information on results incl. tables

The numerical scores awarded to the dermal reactions elicited by the challenge application are shown in Table 1.

The dermal reactions observed in all twenty test animals were more severe than the maximum reaction seen in animals of the control group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
In this test, performed in twenty albino guinea-pigs, DNBP formulation produced evidence of delayed contact hypersensitivity in all twenty animals.
Executive summary:

The potential of the test material, as a ~30% w/v formulation in Mineral Oil, to produce delayed contact hypersensitivity in guinea pigs was evaluated using a Guinea pig maximisation test.

Following primary challenge using the test material, as a 5% v/v formulation in distilled water, all 20 treated animals showed positive responses, compared to 1 of 10 in the negative control group.

DNBP formulation produced evidence of delayed contact hypersensitivity in all twenty animals.