Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 21 October 1997 and 21 November 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to OECD/EU guidelines under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
the recorded relative humidity of the animal room (26-58%) fell below the range given in the protocol (30- 70%), however this was not considered to have had an adverse effect on the animals.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
the recorded relative humidity of the animal room (26-58%) fell below the range given in the protocol (30- 70%), however this was not considered to have had an adverse effect on the animals.
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colourless liquid
Details on test material:
Identity: 97-204-01
Commercial name: Montaverdi
Appearance: Clear colourless liquid
Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
Fifteen healthy male albino guinea-pigs of the Dunkin/Hartley strain were obtained from D. Hall, Newchurch, Staffordshire, England.
The animals were approximately four to seven weeks of age on arrival and were acclimatised to the experimental environment for five days prior to the start of the main study. The guinea-pigs were within the weight range 323 - 423 g at the start of the study (Day 1).
Additional animals from the same supplier were used for the preliminary investigations.
The animals in the main study were allocated without conscious bias to two groups as follows:

Group Number of animals Animal numbers
Control animals 5 4975 to 4979
Test animals 10 4980 to 4989

The guinea-pigs were housed in groups of five in suspended metal cages with wire mesh floors in Building R17 Room 14.
A vitamin C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) and drinking water were provided ad libitum. Hay was given weekly. The batch of diet used for the study was analysed for nutrients, possible contaminants or micro-organisms, likely to be present in the diet, and which, if in excess, may have had an undesirable effect on the test system. The certificates of analyses were lodged in Huntingdon Life Sciences Limited Archives. There
were no known contaminants present in the diet which were expected to be capable of interfering with the study outcome.

Results of routine physical and chemical examination of drinking water, as conducted by the supplier are made available to Huntingdon Life Sciences Ltd as quarterly summaries.

Animal room temperature was controlled within the range 19 to 21 °C and relative humidity within the range 26 to 58%. These environmental parameters were recorded daily. Air exchange was maintained at approximately 15 air changes per hour and lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.

Each animal was identified by ear tattoo number. This number was unique within the Huntingdon Life Sciences Acute Toxicology Department throughout the duration of the study. Each cage was identified by a coloured label displaying the study schedule number, animal numbers and the initials of the Study Director and Home Office licensee.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Alembicol D
Concentration / amount:
Induction intradermal injection - 7.5% v/v in Alembicol D
Induction topical application - as supplied
Topical challenge - as supplied and 50% v/v in Alembicol D
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Alembicol D
Concentration / amount:
Induction intradermal injection - 7.5% v/v in Alembicol D
Induction topical application - as supplied
Topical challenge - as supplied and 50% v/v in Alembicol D
No. of animals per dose:
Test - 10
Control - 5
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 concentrations used are described in the treatment procedure.
The absorption of the test substance was not determined.
# A product of coconut oil, supplied by Alembic Products, Saltney, Chester, England

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 mjection of Freund's complete adjuvant, 50 : 50 with water for irrigation (Ph.Eur.), approximately two weeks prior to the start of the preliminary investigations.
Selection of concentrations of test substance for the main study were based on the results of the preliminary investigations. The following concentrations of Montaverdi were selected:
Induction intradermal injection - 7.5% v/v in Alembicol D
This was the highest concentration that caused irritation but did not adversely affect the animals.
Induction topical application - as supplied
Topical challenge - as supplied and 50% v/v in Alembicol D
From preliminary investigations administration of the test substance as supplied did not give rise to irritating effects.

Main study
The procedure may be considered in two parts, Induction and Challenge.
Induction
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. 97-204-01, 7. 5% v/v in Alembicol D.
3. 97-204-01, 7. 5 % v/v in a 50 : 50 mixture of Freund's complete adjuvant and Alembicol D.

Induction topical application - test animals
The preliminary investigations indicated that the test substance applied topically as supplied (neat) did not produce skin irritation. Therefore, 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 gentle rubbing with 0. 5 ml per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch ofWhatman No. 3 paper was saturated with approximately 0.4 ml of 97-204-01, as supplied. 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.
Induction - control animals
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.
Challenge
Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application using Montaverdi, as supplied and 50% vlv 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 ofWhatman No. 3 paper was saturated with approximately 0.2 ml of 97-204-01, as supplied and applied to an anterior site on the flank. 97-204-01, 50% vlv 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".
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (HCA), benzocaine and 2-mercaptobenzothiazole (MBT).

Results and discussion

Positive control results:
Positive responses were elicited in the animals receiving the positive control articles Hecyl cinnamic aldehyde or 2-Mercaptobenzothiazole.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
as supplied
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: as supplied. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
as supplied
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: as supplied. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
224
Group:
test group
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 224.0. Group: test group. Dose level: 50% v/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50% v/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
as supplied
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: as supplied. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
as supplied
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: as supplied. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50% v/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% v/v in Alembicol D
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50% v/v in Alembicol D. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

CLINICAL SIGNS No signs of ill health or toxicity were recorded. BODYWEIGHT Bodyweight increases were recorded for all guinea-pigs over the period of the study. Intradermal injections Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving Montaverdi, 7.5% 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 Montaverdi, as supplied. Slight erythema was seen in 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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study, Montaverdi did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the ten test animals.
Executive summary:

A GPMT according to OECD406 was performed to assess the skin sensitisation potential of Montaverdi. Dose levels selected for this study were

intradermal injection 7.5% v/v in Alembicol D, topical application as supplied and challenge application as supplied and 50% v/v in Alembicol D. Ten test and five control guinea-pigs were used in this study. Montaverdi did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in any of the ten test animals and therefore does not require labelling with the risk phrase R43 "May cause sensitisation by skin contact" in accordance with the DSD 67/548/EEC or the CLP Regulation (EC) No. 1272/2008.