Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 September 1996 - 25 October 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guideline
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:
Non-LLNA test was also conducted (see Report R&H 241/963705/SS). The results of this guinea pig maximazation test were positive and were therefore chosen as a key study. This study was conducted in 1997.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
MMBC
Alternative name: RH-119, 114 Technical
Appearance: light brown solid
Storage conditions: 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, Staffordshire, England.
- Age at arrival: 4 - 5 weeks.
- Weight on arrival: 313 - 368 g.
- Housing: groups of five in suspended metal cages with wire mesh floors.
- Diet (e.g. ad libitum): A vitamin C enriched guinea-pig diet FD2 was provided ad libitum. Hay was given weekly.
- Water (e.g. ad libitum): water was provided ad libitum.
- Acclimation period: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 - 20.5 °C.
- Humidity (%): 43 - 62% relative.
- Air changes (per hr): approximately 15 per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: intrandermal and topical
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
Induction intradermal injection: 1 % w/v in Alembicol D and 1 % w/v in a 50:50 mixture of Freund's Complete adjuvant and Alembicol D.

Induction topical application: 7.5 % w/v in Alembicol D.

Topical challnge: 5 and 2.5 % w/v in Alembicol D.
Challengeopen allclose all
Route:
other: topical
Vehicle:
other: Alembicol D (a product of coconut oil)
Concentration / amount:
Induction intradermal injection: 1 % w/v in Alembicol D and 1 % w/v in a 50:50 mixture of Freund's Complete adjuvant and Alembicol D.

Induction topical application: 7.5 % w/v in Alembicol D.

Topical challnge: 5 and 2.5 % w/v in Alembicol D.
No. of animals per dose:
10
Details on study design:
TEST SUBSTANCE PREPARATION
The test substance was prepared prior to each application on the day of dosing in Alembicol D.
The absorption of the test substance was not determined.
The homogeneity, stability and purity of the test substance in the solvents were not determined as part of this study.

TREATMENT PROCEDURE
Preliminary study

The intradermal and topical irritancy of a range of dilutions 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 irrigation (Ph.Eur.), approximately one or two weeks prior to the start of the preliminary investigations.
Based on the results of the preliminary investigations, the following concentrations of MMBC were selected:

Induction intradermal injection - 1 % w/v in Alembicol D
This was the highest concentration that caused irritation but did not adversely affect the animals.

Induction topical application - 7.5 % w/v in Alembicol D
This was the highest concentration that produced some irritation but did not adversely affect the animals.

Topical challenge - 5 and 2.5 % w/v in Alembicol D
From preliminary investigations 5 % w/v in Alembicol D was the highest concentration not giving rise to irritating effects.


MAIN STUDY
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.
2. MMBC, 1 % w/v in Alembicol D.
3. MMBC, 1 % w/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D.

Topical application - test animals

One week after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair.
A 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 mL of MMBC, 7.5 % 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 round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.

During the induction phase, 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.
The dermal reactions were observed after each induction phase in both control and test animals.

CHALLENGE
Challenge - control and test animals

The control and test animals were challenged topically two weeks after the topical induction application using MMBC 5 and 2.5 % 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 MMBC, 5 % w/v in Alembicol D and applied to an anterior site on the flank. MMBC, 2.5 % w/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".

OBSERVATIONS
Clinical signs
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 applications.

Dermal responses
The dermal reactions resulting from intradermal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system:

Erythema and eschar formation:
No erythema 0
Slight erythema 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 1
Well-defined oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately I millimetre) 3
Severe oedema (raised more than 1 millimetre and extending 4
beyond the area of exposure)

The approximate diameter (mm) of the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study.

Any other lesion not covered by this scoring system was described.

The challenge sites were evaluated 24 and 48 hours after removal of the patches.

INTERPRETATION OF RESULTS
Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals.

A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group.

If the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive.

A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than, the maximum reaction seen in animals of the control group.
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically with known sensitisers hexyl cinnamic aldehyde (HCA), Benzocaine and mercaptobenzothiazole (MBT).

Results and discussion

Positive control results:
The positive controls were valid.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: Challenge Application
Hours after challenge:
24
Group:
test group
Dose level:
2.5 and 5 % w/v test material in Alembicol D
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: other: Challenge Application. . Hours after challenge: 24.0. Group: test group. Dose level: 2.5 and 5 % w/v test material in Alembicol D. No with. + reactions: 10.0. Total no. in groups: 10.0.
Reading:
other: Challenge Application
Hours after challenge:
48
Group:
test group
Dose level:
2.5 and 5 % w/v test material in Alembicol D
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: other: Challenge Application. . Hours after challenge: 48.0. Group: test group. Dose level: 2.5 and 5 % w/v test material in Alembicol D. No with. + reactions: 10.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Table 1 Dermal Results Observed After Each Induction

Site

Intradermal injection

Topical Application

Test animals

Control animals

Test animals

Control animals

1

Necrosis

Necrosis

Moderate erythema

No

erythema

2

Slight irritation

Slight irritation

3

Necrosis

Necrosis

Control animals:

1) 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation.

2) 0.1 mL of Alembicol D.

3) 0.1 mL of Freund's complete adjuvant 50:50 with Alembicol D.

Test animals:

1) 0.1 mL of Freund's complete adjuvant 50:50 with water for irrigation.

2) 0.1 ml of MMBC 1 % w/v in Alembicol D.

3) 0.1 ml of MMBC 1 % w/v in a 50:50 mixture of Alembicol D and Freund's complete adjuvant.

 

Table 2 Dermal Reactions observed After the Challenge Application

Individual Reactions for Test Animals

 

Score

 

Results

24 hours

48 hours

A

P

A

P

Erythema

Oedema

1

1

2

1

2

1

2

1

+

Erythema

Oedema

2*

2

2

1

2

3*

2**

1

+

Erythema

Oedema

1

1

1

1

1

0

1

0

+

Erythema

Oedema

2

2

2

1

2

2

2

1

+

Erythema

Oedema

2

1

1

1

1

1

1

1

+

Erythema

Oedema

1

1

1

0

1

1

1

1

+

Erythema

Oedema

2

2

2

1

2

2

2

2

+

Erythema

Oedema

2

1

1

1

1

1

1

0

+

Erythema

Oedema

2

1

2

1

2

2

2

1

+

Erythema

Oedema

2

1

1

1

2

1

2

1

+

A = Anterior site, exposed to MMBC 5 % w/v in Alembicol D

P = Posterior site, exposed to MMBC 2.5 % w/v in Alembicol D

*Necrotic patch

**Necrotic Edge

Thickening, dryness and sloughing of the epidermis

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study the test substance produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten test animals.
Executive summary:

The skin sensitisation potential of the test substance was determined in accordance with standardised guideline EU Method B.6. In this study the test substance produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten test animals. The positive controls were shown to have the capacity to cause skin sensitisation confirming the validity if the protocol used.