Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd. Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf / Switzerland
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: (pre-test group) Males: 356 - 398 g Females: 304 - 351 g
(control and test groups( Males: 316 - 371 g Females: 298 - 402 g
- Housing: Individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 22.5°C
- Humidity: 47 - 73 %
- Air changes: 10 - 15/hour
- Photoperiod: 12 hours light/12 hours datk

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
peanut oil
Concentration / amount:
Intradermal induction (day 1): 5 %
Epidermal induction (day 8): 100 %
Epidermal application (day 18): 0.5 %
Challengeopen allclose all
Route:
epicutaneous, semiocclusive
Vehicle:
peanut oil
Concentration / amount:
Intradermal induction (day 1): 5 %
Epidermal induction (day 8): 100 %
Epidermal application (day 18): 0.5 %
No. of animals per dose:
Control group: 5 Male, 5 Female
Test group: 10 Male, 10 Female
Details on study design:
RANGE FINDING TESTS:
PRE-TESTS
The intradermal and epidermal pre-test I were performed with the test material before and during acclimatization period of the control and test group.
The epidermal pre-test II was performed during the intradermal and epidermal induction of the control and test group.
The epidermal pre-test III was performed after the epidermal induction of the control and test group.
The second and third pre- tests (to determine the concentration for epidermal challenge) were necessary due to findings of irritation at successively lower concentrations in the epidermal pre-test I.

PRE-TESTS - INTRADERMAL INJECTIONS
Four intradermal injections (0.1ml/site) of a 1:1 (v/v) mixture of FCA/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5%, 3% and 1%. of the test item in the vehicle.
Dermal reactions were assessed after a 24 hour period.

EPIDERMAL APPLICATION – EPIDERMAL PRE-TEST I
Four intradermal injections (0.1ml/site) of a 1:1 (v/v) mixture of FCA/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application.
Thereafter 4 patches of filter paper (3x3cm) were saturated with the test item at 1005, 75%, 50% and 25% and applied to the clipped and shaved flanks. Vehicle was used for dilution. The volume of test item preparation applied was approximately 0.2ml. The patches were covered by a strip of aluminium foil and firmly secured by plastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test item.
The dressings were removed after an exposure period of 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.


EPIDERMAL APPLICATION – EPIDERMAL PRE-TEST II
As no highest non-irritating concentration could be determined after the epidermal pre-test, a second epidermal pre-test was performed with two additional naïve guinea pigs. The animals were pre-treated with FCA/saline and treated in the same way as those described previously, with the concentrations of 15%, 10%, 5% and 1% in vehicle, also using an application of approximately 0.2ml.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.

EPIDERMAL APPLICATION – EPIDERMAL PRE-TEST III
As no highest non-irritating concentration was determined in the second epidermal pre-teat, a third epidermal pre-test was conducted with two additional naïve guinea pigs pre-treated with FCA/saline as previously (only five days between pre-treatment and treatment) and treated epidermally with the concentrations of 0.5%, 0.1%, 0.05% and 0.01% in vehicle. An application volume of approximately 0.2ml was used.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.

SELECTION OF CONCENTRATIONS FOR THE MAIN STUDY:
INTRADERMAL INDUCTION
The 5% concentration of the test item selected for the induction exposure was well tolerated systemically and caused mild to moderate skin irritation. Higher concentrations were not tested because the 5% concentration is the maximum concentration for intradermal induction recommended.

EPIDERMAL INDUCTION
The 100% concentration of the test item used for the induction exposure was the highest concentration Causing mild to moderate skin irritation. It was well tolerated systemically.

EPIDEMAL CHALLENGE
The 0.5% concentration of the test item used for the challenge application was the highest tested non-irritant concentration.

From the observed findings the test item at 100% was considered to be the tolerated concentration to stimulate a state of immune hypersensitivity and 0.5% as the highest non-irritating concentration to be used for the challenge.


MAIN STUDY
A. INDUCTION EXPOSURE
INDUCTION
INTRADERMAL INJECTIONS – TEST DAY 1
An area of dorsal skin from the scapular region (approx. 6x8cm) was clipped free of hair. Three pairs of intradermal injections (0.1ml/site) were made at the border of a 4x4cm area clipped region as follows:
Test group
1) 1:1 (v/v) mixture of FCA and physiological saline.
2) The test item at 5 % in the vehicle
3) The test item at 5% in a 1:1 (v/v) mixture of FCA and physiological saline.
Control group
1) 1:1 (v/v) mixture of FCA/physiological saline.
2) The vehicle alone.
3) 1:1 (w/w) mixture of the vehicle and a 1:1 (v/v) mixture of FCA and physiological saline.

EPIDERMAL APPLICATIONS – TEST DAY 8
On test day 8, a 2x4cm patch of filter paper was saturated with the undiluted test item and placed on the shaved area over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3ml. The patch was covered with aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place for 48 hours. This procedure ensures intensive contact of the test item.
The guinea pigs of the control group were treated as described above with the vehicle only, also applied at a volume of approximately 0.3ml.


B. CHALLENGE EXPOSURE
CHALLENGE – TEST DAY 25
The test and control guinea pigs were challenged 17 days after the epidermal induction application and were treated in the same way.
Hair was clipped and shaved from a 5x5cm area on the left and right flank of each guinea pig just prior to the application. Two patches (3x3cm) of filter paper were saturated with the test item at the highest tested non-irritating concentration of 0.5% (applied to the left flank) and the vehicle only (applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle applied was approximately 0.2ml.
The dressings were left in place for 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema.

Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde

Results and discussion

Positive control results:

For results refer to Table 4.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.5%
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: 0.5%. No with. + reactions: 20.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.5%
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: 0.5%. No with. + reactions: 20.0. Total no. in groups: 20.0.

Any other information on results incl. tables

Results for the test item 

Skin Reactions after the Challenge Procedure

 

 

After 24 hours

After 48 hours

 

 

  Positive / Total__

% Positive of Total

 

 

  Positive / Total__

% Positive of Total

 

Control Group

Test Item, 0.5 % in vehicle

(left flank)

0 / 10

0

0 / 10

0

Vehicle only

(right flank)

0 / 10

0

0 / 10

0

Test Group

Test Item, 0.5 % in vehicle

(left flank)

20 / 20

100

20 / 20

100

Vehicle only

(right flank)

0 / 20

0

0 / 20

0

 

 

Table 4

Positive Control – Skin Reactions after the Challenge Procedure

 

After 24 hours

After 48 hours

 

 

  Positive / Total__

% Positive of Total

 

 

  Positive / Total__

% Positive of Total

 

Control Group

Alpha-hexylcinnamaldehyde, 1 % in PEG 300

(left flank)

0 / 5

0

0 / 5

0

PEG 300 only

(right flank)

0 / 5

0

0 / 5

0

Test Group

Alpha-hexylcinnamaldehyde, 1 % in PEG 300

(left flank)

7 / 10

70

5 / 10

50

PEG 300 only

(right flank)

0 / 10

0

0 / 10

0

 

Applicant's summary and conclusion

Interpretation of results:
other: May cause sensitization by skin contact
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Conclusion

The 100 % incidence of challenge skin reactions to the test item is above the threshold (30 % for adjuvant test) set in Commission Directive 96/54/EEC. Therefore, CA 2814 B (Intermediate of CGA 276854) is required to be classified as ‘May cause sensitization by skin contact’.
Executive summary:

 

In order to assess the cutaneous allergenic potential of CA 2814 B (Intermediate of CGA 276854), the Maximisation-Test was performed in 5 male and 5 female control and 10 male and 10 female test albino guinea pigs, in accordance with OECD Guideline NO. 406 and Directive 96/54/EEC, B.6.

 

The intradermal induction of the test group animals was performed with 5 % concentrations of the test item in the vehicle (peanut oil) and in Freund’s Complete Adjuvant (FCA) (as 1:1 FCA in physiological saline). Control group animals were injected with the vehicle alone and FCA/physiological saline alone. Beginning on study day 8, the epidermal induction was conducted for 48 hours with 100 % concentration of the test item (test group animals) or vehicle alone (control group animals). Seventeen days after epidermal induction, the control and test animals were challenged by epidermal application of the test item at 0.5 % in the vehicle and the vehicle alone. Cutaneous reactions evaluated 24 and 48 hours after removal of the dressing. 

 

Results

Skin Reactions after the Challenge Procedure

 

 

After 24 hours

After 48 hours

 

 

  Positive / Total__

% Positive of Total

 

 

  Positive / Total__

% Positive of Total

 

Control Group

Test Item, 0.5 % in vehicle

(left flank)

0 / 10

0

0 / 10

0

Vehicle only

(right flank)

0 / 10

0

0 / 10

0

Test Group

Test Item, 0.5 % in vehicle

(left flank)

20 / 20

100

20 / 20

100

Vehicle only

(right flank)

0 / 20

0

0 / 20

0

 

All test animals showed discrete/patchy to moderate/confluent erythema at the 24- and 48-hour reading after the challenge treatment with CA 2814 B (Intermediate of GCA 276854) at 0.5 % (w/w) in the vehicle. No skin effect was observed in the control group. 

 

No toxic symptoms were evident in the guinea pigs of the control or test group. 

 

No deaths occurred. 

Conclusion

 

The 100 % incidence of challenge skin reactions to the test item is above the threshold (30 % for adjuvant test) set in Commission Directive 96/54/EEC. Therefore, CA 2814 B (Intermediate of CGA 276854) is required to be classified as ‘May cause sensitization by skin contact’.