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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was conducted in 1998, prior to the introduction of 1907/2006/EC

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identity: CP-I63,625-BV
Chemical name: Not supplied
Intended use: Pharmaceutical intermediate
Appearance: White powder
Storage conditions: Room temperature in the dark
Lot No: 38654-187-3
Purity: 99%

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Fifteen healthy female (which were nulliparous and non-pregnant) albino guinea-pigs of the Dunkin/Hartley
strain were obtained from D. Hall, Newchurch, Staffs, UK.
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 329 - 424 g at the start of the study (Day 1).
Additional animals from the same supplier were used for the preliminary investigations.
The animals on the main study were allocated without conscious bias to two groups as follows:

Group Number of Animal
animals numbers
Control animals 5 2155 to 2159
Test animals 10 2160 to 2169

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 thrice 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 18 to 27°C and relative humidity within the range
47 to 69%. 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
Vehicle:
coconut oil
Concentration / amount:
1. Freund's complete adjuvant** was diluted with an equal volume of water for irrigation
(Ph.Eur.).
CP-163,625-BV, 1% w/v in Alembicol D*.
CP-163,625-BV, 1% w/v in a 50 : 50 mixture of Freund's complete adjuvant and
Alembicol D.
*A product of coconut oil, supplied by Alembic Products, Saltney, Chester, England
Day(s)/duration:
Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
coconut oil
Concentration / amount:
50% w/v in Alembicol D
Day(s)/duration:
48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
coconut oil
Concentration / amount:
50 and 25% w/v in Alembicol D
Day(s)/duration:
24 and 48 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10
Details on study design:
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 week prior 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 CP-163,625-BV were
selected:
Induction intradermal injection - 1% w/v in Alembicol D

This was the maximum practical concentration for intradermal administration and caused irritation
but did not adversely affect the animals.

Induction topical application - 50% w/v in Alembicol D
Topical challenge - 50 and 25% w/v in Alembicol D

From preliminary investigations 50% w/v in Alembicol D was the maximum practical concentration
that could be prepared and dosed topically and 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. CP-163,625-BV, 1% w/v in Alembicol D.
3. CP-163,625-BV, 1% w/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 maximum practical concentration of the test
substance for topical application (50%) 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 of Whatman No. 3 paper was saturated
with approximately 0.4 ml of CP-163,625-BV, 50% 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.

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 CP-163,625-BV, 50 and 25% 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
CP-163,625-BV, 50% w/v in Alembicol D and applied to an anterior site on the flank. CP-163,625-
BV, 25% 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 "Blenderrn" covered by "Elastoplast" wound round
the trunk and secured with "Sleek".

Results and discussion

Positive control results:
The sensitivity of the guinea-pig strain used is checked periodically at Huntingdon Life Sciences with
known sensitisers hexyl cinnamic aldehyde (HCA), Benzocaine and 2-mercaptobenzothiazole (MBT).
Grade 1/2 edema and erythema in all 10 animals treated with 50% w/v and 100% w/v HCA (10/10 positive)

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 % w/v
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 % w/v
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of ill health or toxicity were recorded.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100% w/v
No. with + reactions:
10
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100% w/v
No. with + reactions:
10
Total no. in group:
10

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. There were no remarkable findings.

INDUCTION

Intradermal injections

Necrosis was recorded at most sites receiving Freund's Complete Adjuvant in test and control animals. Slight to well-defined irritation was seen in test animals at sites receiving CP-163,625-BV, 1% w/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D.

Topical application

Slight to well-defined erythema was observed in test animals following topical application with CP-163,625-BV, 50% w/v in Alembicol D. Slight erythema was seen in the control guinea-pigs.

CHALLENGE

There were no dermal reactions seen in any of the test or control animals that were considered to be related to treatment, therefore all ten test animals gave negative responses. Dryness and sloughing of the epidermis was noted for one control and two test animals, however, this was considered to represent background irritation.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study, CP-163,625-BV did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals.
Executive summary:

This study was performed to assess the skin sensitization potential of CP-163,625-BV using the guinea-pig. The method followed was that described in:

EEC Methods for the determination of toxicity, Annex to Directive 96/54/EEC (Official Journal No. L248, 30.9.96), Part B, Method B.6. Skin sensitization;

OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted: 17 July 1992.

MAGNUSSON, B. And KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig: Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A.

The guinea pigs were dosed by intradermal injection and topical application as these are the routes of exposure required by the test guideline and method. Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected:

Intradermal injection: 1% w/v in Alembicol D

Topical application: 50% w/v in Alembicol D

Challenge application: 50 and 25% w/v in Alembicol D

Ten test and five control guinea-pigs were used in this study. In this study CP-163,625-BV did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. CP-163,625-BV does not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Commission Directive 93/21/EEC.