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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
The relative humidity was observed below 40 % in the test laboratory
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:
other: Himalayan Spotted (GOH)
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: Pretest groups - males 362 - 368 g, females 358 g. Control and test group - males 317 - 406 g, females 294 - 336 g.
- Housing: Individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ° C
- Humidity (%): 28 - 55 %
- Air changes (per hr): 10 - 15/hour
- Photoperiod (hrs dark / hrs light): 12/12/hours

The animal were distributed as follows:

Number of animals per group Animal numbers per group
1 Control Group 10 422 - 431
2 Test Group 20 432 - 451
3 Intradermal Pre-test 1 452
4 Epidermal Pre-test 2 453 - 454


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % + 1 % Tween 80 in distilled water
Concentration / amount:
Intradermal Induction: 5 %
Epidermal Induction: 75 %
Epidermal Challenge: 75 %
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1 % + 1 % Tween 80 in distilled water
Concentration / amount:
Intradermal Induction: 5 %
Epidermal Induction: 75 %
Epidermal Challenge: 75 %
No. of animals per dose:

Number of animals per group Animal numbers per group
1 Control Group 10 422 - 431
2 Test Group 20 432 - 451
3 Intradermal Pre-test 1 452
4 Epidermal Pre-test 2 453 - 454


Details on study design:
RANGE FINDING TESTS:
Pre-test:
The pre-tests were performed with the test material before and during the acclimatization period of the control and test group.
Intradermal Injections:
The three concentrations for the intradermal injection pre-tests were determined during formulation trials performed prior to the acclimatization. The concentration of 5 % was considered to be the highest technically applicable concentration which could be injected into the intra-cellular space in spite of the high viscosity of the application dilution and the obstacle caused by the tissues.
Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of FVA/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/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 24 hours later using the Magnusson and Klingman scoring method.

Epidermal injections:
Prior to testing, it was determined that a 75 % concentration of the test material in the vehicle was the highest which could be applied to the skin to ensure optimal skin contact during the treatment period and to avoid mechanical irritation caused by the test item.
Four intradermal injections 0.1 ml/site of a 1:1 (v/v) mixture of FCA/physiological saline were made to 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 (3 x 3 cm) were saturated with the test item at 75, 50, 25 and 15 % in the vehicle and applied to the clipped and shaved flanks. The amount of test item preparation applied was approximately 0.2 g for the test item at 75 % and 50 % and a volume of approximately 0.2 ml was applied for the remaining test item concentrations. The patches were covered by a strip of aluminium foil and firmly secured by elastic 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 of 24 hours.
The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Klingman.


Main Study

Intradermal injections - Test Day 1:
3 x 0.1 ml injections
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 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 – Day 8:
Test group
Approximately 0.3 g of the test item (75 % in vehicle) was saturated on a 2 x 4 cm patch of filter paper.

Control group
0.3 ml of the vehicle only.

Challenge – Day 22
Test and Control group were treated in the same way.
Left and right flank of each animal clipped and shaved.
2 patches of 3 x 3 cm filter paper were saturated with 0.2 g of the test item at the highest non-irritating concentration of 75 % and 0.2 ml of vehicle
only. These patches were applied to and applied to the left flank (test item) and the right flank (vehicle), using the same method as for the epidermal application.
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: Epdermal Induction
Hours after challenge:
24
Group:
other: Control
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Discrete/patchy erythema
Remarks on result:
other: Reading:
Reading:
other: Epidermal Induction
Hours after challenge:
48
Group:
other: Control
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
Discrete/patchy erythema
Remarks on result:
other: Reading:
Reading:
other: Epidermal Induction
Hours after challenge:
24
Group:
test group
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
other: Reading:
Reading:
other: Epidermal Induction
Hours after challenge:
48
Group:
test group
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
other: Reading:
Reading:
other: Challenge
Hours after challenge:
24
Group:
other: Control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reaction observed
Remarks on result:
other: Reading:
Reading:
other: Challenge
Hours after challenge:
48
Group:
other: Control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reaction observed
Remarks on result:
other: Reading:
Reading:
other: Challenge
Hours after challenge:
24
Group:
test group
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
Discrete/patchy to moderate/conflent erythema
Remarks on result:
other: Reading:
Reading:
other: Challenge
Hours after challenge:
48
Group:
test group
No. with + reactions:
16
Total no. in group:
20
Clinical observations:
Dicrete/patchy to moderate/confluent erythema
Remarks on result:
other: Reading:

Any other information on results incl. tables

There were no deaths during the course of the study, hence no necropsies were performed. 

No signs of systemic toxic were observed in the animals. 

The body weight of the animals was within range commonly recorded for animals of this strain and age. 

Applicant's summary and conclusion

Interpretation of results:
other: May cause sensitization by skin contact
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The 80 % incidence of skin reactions to the test material is above the threshold (30 % for adjuvant tests) set in Commission Directive 93/21/EEC. Therefore, CA 2218 A (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 2218 A (Intermediate of CGA 276854), 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 the 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 (1 % CMC + 1 % Tween 80 in distilled water) and in Freund’s Complete Adjuvant (FCA) (as 1:1 FCA in physiological saline). Control group animals were injected with the vehicle control alone and FCA/physiological saline alone. On study day 7, the test areas of all animals were pre-treated with 10 % sodium-lauryl-sulfate. Beginning on study day 8, the epidermal induction was conducted for 48 hours with a 75 % concentration of the test item in the vehicle (test group animals) or vehicle alone (control group animals). Two weeks after epidermal induction, the control and test animals were challenged by epidermal application of the test item at 75 % in the vehicle and the vehicle alone. Cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing. 

 

After 24 hours

 

Positive / Total

 

% positive of total

After 48 hours

 

Positive / Total

 

% positive of total

CONTROL GROUP

 

CA 2218 A, 75 % in vehicle (left flank)

 

 

Vehicle only

 

 

 

0 / 10

0

 

0 / 10

0

 

 

0 / 10

0

 

0 / 10

0

TEST GROUP

 

CA 2218 A, 75 % in vehicle (left flank)

 

 

Vehicle only

 

 

 

15 / 20

75

 

0 / 20

0

 

 

16 / 20

80

 

0 / 20

0

 

Fifteen (at the 48 hour reading) and 16 (at the 48 hour reading) out of the 20 test animals showed discrete/patchy to moderate/confluent erythema after challenge treatment with CA 2218 A (Intermediate of CGA 276854) at 75 % (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. 

 

The 80 % incidence of skin reactions to the test material is above the threshold (30 % for adjuvant tests) set in Commission Directive 93/21/EEC. Therefore, CA 2218 A (Intermediate of CGA 276854) is required to be classified as ‘May cause sensitization by skin contact’.