Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May to 24 June, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to EU method B.6. GLP study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): OS 2200 (489-95A).
- Physical state: Clear to light yellow liquid.
- Analytical purity: >92%
- Lot/batch No.:38659-14
- Expiration date of the lot/batch: 1 January 1996
- Storage condition of test material: Room temperature in dark under nitrogen.

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 study initiation:4 to 5 weeks.
- Weight at study initiation: 279-338g.
- Housing: Groups of five were housed in suspended metal cages with mesh floors.
- Diet: Ad libitum. Vitamin C enriched guinea-pig diet FD2. Hay was given weekly.
- Water: Ad libitum.
- Acclimation period: 5 days prior to allocation to the main study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21ºC.
- Humidity: 30-70 %.
- Air changes: 15 per hour.
- Photoperiod: 12hours dark/12 hours artificial light.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: acetone
Concentration / amount:
Intradermal induction: 7.5% in acetone 5% in alembicol d.
topical induction: as supplied.
topical challenge: as supplied and 50% v/v in acetone.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
Intradermal induction: 7.5% in acetone 5% in alembicol d.
topical induction: as supplied.
topical challenge: as supplied and 50% v/v in acetone.
No. of animals per dose:
10
Details on study design:
RAGE FINDING TESTS:
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to determine concentrations that would produce irritation suitable for the induction phase of the main study and the maximum non-irritant concentration by the topical route of administration for the challenge phase. 7.5% v/v in 5% acetone in Alembicol D was the highest concentration that caused irritation but did not adversely affect the animals. The test substance applied topically as supplied did not give rise to irritating effects.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal (three pairs of injections) and 1 dermal.
- Exposure period: not applicable (intradermal), 48 hours (dermal).
- Test groups: Test substance, test substance + FCA (Freund's Complete adjuvant), FCA.
- Control group: FCA, FCA+ vehicle, vehicle.
- Site: A 40x60 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 20x40 mm area.
- Frequency of applications: intradermal on day 0, dermal at day 7
- Duration: 7 days
- Concentrations: Intradermal induction (three pair of injections):
Test substance: 7.5% v/v in 5% acetone in Alembicol D
Test substance + FCA: 7.5% v/v in a 50:50 mixture of FCA and 5% acetone in Alembicol D
FCA: diluted with an equal volume of water .

Six days after the injections the site was pre-treated by gentle rubbing with 0.5 ml per site of 10% w/w sodium lauryl sulphate in petrolatum. 24 hours later a 20 x 40 mm patch of paper was saturated with approximately 0.4 ml test substance as supplied. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This was firmly secured by elastic adhesive bandage wound round the torso of the animal and fixed with impervious plastic adhesive tape. The dressing was left in place for 48 hours.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 21 (two weeks after induction)
- Exposure period: 24h
- Test groups: Test substance
- Control group: Test substance
- Site: 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 paper was saturated with approximately 0.2 ml of test substance as supplied and applied to an anterior site on the flank. 50% v/v of the test substance in acetone was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips covered by wound round the trunk and secured.
- Concentrations: as supplied and 50% v/v in acetone
- Evaluation (h after challenge): 24, 48, 72 h after the removal of the patches

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 application.
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 Draize scoring system.
Challenge controls:
0.1mL of 5% acetone in Alembicol D.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde

Results and discussion

Positive control results:
The positive controls are periodically checked by Huntingdon Life Sciences to detect skin sensitization potential. The last study was performed between 8 December 1992 and 18 February 1995. In this study HCA produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten animals, thus confirming the sensitivity and reliability of the experimental technique.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 7.5%. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 7.5%. Challenge: As supplied.. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no erythema, no oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 7.5%. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 7.5%. Challenge: As supplied.. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no erythema, no oedema.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
Induction: 7.5%. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading:
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 7.5%. Challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Induction: 7.5%. Challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no erythema, no oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 7.5%. Challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Induction: 7.5%. Challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no erythema, no oedema.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
Induction: 7.5%. Challenge: 50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading:
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: 5% acetone. Challenge: As supplied.. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no erythema, no oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: 5% acetone. Challenge: As supplied.. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no erythema, no oedema.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: As supplied.
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading:
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Induction: 5% acetone. Challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no erythema, no oedema.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Induction: 5% acetone. Challenge: 50%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no erythema, no oedema.
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
Induction: 5% acetone. Challenge: 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no erythema, no oedema
Remarks on result:
other: Reading:

Any other information on results incl. tables

CLlNICAL 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.

INDUCTION

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 7.5% v/v in 5% acetone in Alembicol D and slight irritation was observed in control animals receiving 5% acetone in Alembicol D alone.

Topical application:

Slight erythema was observed in test animals following topical application with test item as supplied and slight erythema was seen in the control guinea-pigs.

CHALLENGE

There were no dermal reactions seen in any of the test or control animals:

Table 1. Dermal reactions observed after the challenge application with OS-2200

Guinea-pig number

E= Erythema

O= Oedema

Score

Results

Positive+
Negative -
Inconclusive +/-

24 Hours

48 Hours

72 Hours

A

P

A

P

A

P

Freund’s treated controls

 

1855

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

1856

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

1857

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

1858

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

1859

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

Test animals

 

1860

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1861

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1862

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1863

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1864

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1865

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1866

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1867

E

0

0

0

0

0

0

-

 

O

0

0

0

0

0

0

1868

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

1869

E

0

0

0

0

0

0

-

O

0

0

0

0

0

0

A: Anterior site, exposed to OS-2200 as supplied.
P: Posterior site, exposed to OS-2200, 50% v/v in acetone.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In the Guinea Pig Maximisation Test, OS-2200 did not produce evidence of skin sensitisation in any of the ten test animal.
Executive summary:

A skin sensitisation study was performed with the substance OS-2200 according to EU method B.6 under GLP conditions. A group of 10 guinea-pigs were intradermally (OS-2200 7,5% v/v in 5% acetone in alembicol d) and topically (six days after injections, as supplied) induced to develop an immune-hypersensitive state, during this phase a group of 5 guinea-pigs were treated similarly to the test animals with the exception that the test substance was omitted from intradermal injection and topical application. Sensitisation is potentiated by the injection of Freund's complete adjuvant, validity criteria were fulfilled. Two weeks after the topical induction animals were topically challenged using patches of OS-2200 as supplied and 50% v/v in acetone for 24 hours. All animals were observed daily and no signs of ill health or toxicity were recorded, the challenge sites were evaluated 24, 48 and 72 hours after removal of the patches and there were no dermal reactions seen in any of the test or control animals. In the study, OS-2200 did not produce evidence of skin sensitisation.