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Administrative data

Description of key information

The potential of the substance to induce delayed contact hypersensitivity was investigated using the Maximization method of Magnusson and Kligman (OECD 406, GLP). The result of the Guinea Pig Maximisation Test indicates that the substance was a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2000-01-25 to 2000-10-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study predates LLNA REACH requirements
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffs, UK
- Age at study initiation: 4 to 7 weeks
- Weight at study initiation: 351 - 428 g
- Housing: in groups of five suspended in metal cages with mesh floors
- Diet (e.g. ad libitum): A vitamin C enriched guinea-pig diet ad libitum ; hay was given thrice weekly
- Water (e.g. ad libitum): water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.5 to 23.0°C
- Humidity (%): 39 to 63%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): a switch time to give 12 hours of artificial light (0600-1800 hours GMP) in each 24 hours period


Route:
intradermal and epicutaneous
Vehicle:
vegetable oil
Remarks:
Commercial name: Alembicol D ( coconut oil supplied by Alembic products, Saltney, Chester, England)
Concentration / amount:
Concentration of test material and vehicle used at induction (intradermal and topical exposure):
Intradermal injection : 2.5% w/v* in Alembicol D Topical application : 60% w/v* in Alembicol D
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Concentration of test material and vehicle used for challenge: 20% ** and 10% w/v in Alembicol D
** highest non-irritant concentration based on preliminary assays
Route:
epicutaneous, occlusive
Vehicle:
vegetable oil
Remarks:
Commercial name: Alembicol D ( coconut oil supplied by Alembic products, Saltney, Chester, England)
Concentration / amount:
Concentration of test material and vehicle used at induction (intradermal and topical exposure):
Intradermal injection : 2.5% w/v* in Alembicol D Topical application : 60% w/v* in Alembicol D
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Concentration of test material and vehicle used for challenge: 20% ** and 10% w/v in Alembicol D
** highest non-irritant concentration based on preliminary assays
No. of animals per dose:
- preliminary test: 4 treated animals
- main test: 5 control animals and 10 treated animals
Details on study design:
RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study

-Intradermal exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Intradermal administration of 0.1 ml of the test material (TM) at concentrations from 0.1 to 10% w/v in 2 animals.
Evaluation of the potential cutaneous reactions: 24 and 72 hours after injection

- Epicutaneous exposure:
Hair over the flanks were removed using electric clippers before treatment.
Each selected concentration applied to a filter paper patch measuring 4cm2 for 24 hours under occlusive dressing . 4 concentrations (60, 40, 20 and 10% w/v in Alembicol D) were tested in 4 animals.
Evaluation of the potential cutaneous reactions 24 and 48 hours after patch removal.

MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. TM at 2.5 % w/v in vehicle
. TM at 2.5% w/v in a 50/50 (v/v) mixture of FCA and vehicle
Epicutaneous exposure
Application of 8 cm2 patch saturated with the undiluted TM to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. vehicle
. FCA diluted to 50% with vehicle

Epicutaneous exposure
Application of 8 cm2 patch saturated with the vehicle to the scapular region and held in place for 48 hours using an occlusive dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (2x 4cm) in the scapular region

Epicutaneous exposure
2x 4cm in the interscapular area

- Frequency of applications:
One intradermal injection and one epicutaneous application on Day 8 on the same site.

- Duration:
Epicutaneous exposure: 48 hours

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours

-> TEST GROUPS:
Challenge: TM at 20% in the vehicle on an anterior site of the left flank and TM at 10% in the vehicle on the posterior site of the same flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group

- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize.
Challenge controls:
Control animals received vehicle only.
Positive control substance(s):
yes
Remarks:
hexyl cinnamic aldehyde (HCA)
Positive control results:
The positive control study was conducted between 15 december 1999 and 8 january 2000 using Hexyl cinnamic aldehyde (HCA).
Slight to well-defined dermal reactions were observed for nine of the ten animals comparing to the no dermal reactions in the control animals. Therefore
these nine test animals gave positive sensitization responses.
In this study, HCA produced evidence of skin sensitisation in nine of ten animals, thus confirming the sensitivity and reliability of the experimental
technique.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20 %
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Slight erythema
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
Slight erythema
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Slight to well-defined erythema with or without slight oedema
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Slight to well-defined erythema with or without slight oedema
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20 %
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20 %
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
Dryness and sloughing of the epidermis
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

7.4.1.2: Dermal reactions observed after the challenge application:

Group

Animal number

E=Erythema

O =Oedema

Skin reaction (hour after removal of dressing)

Results

Positive (+)

Negative (-)

Inconclusive (±)

24 hours

48 hours

A

P

A

P

Control group

1990

E

0

0

1*

0

 

O

0

0

0

0

1991

E

0

0

0

0

O

0

0

0

0

1992

E

0

0

0

0

O

0

0

0

0

1993

E

0

0

0

0

O

0

0

0

0

1994

E

0

0

0

0

O

0

0

0

0

Treated group

1995

E

1

1

1

1

+

O

0

0

1

1

1996

E

0

0

0

0

-

O

0

0

0

0

1997

E

1

1

2

2

+

O

0

0

1

1

1998

E

0

0

0

0

-

O

0

0

0

0

1999

E

0

0

0

1

-

O

0

0

0

0

2000

E

0

0

2Td

2*

+

O

0

0

1

1

2001

E

1

1

2

2

+

O

0

0

1

1

2002

E

0

0

0

0

-

O

0

0

0

0

2003

E

0

0

1

0

-

O

0

0

0

0

2004

E

1

1

1

2

+

O

0

0

0

1

* Dryness and sloughing of the epidermis

Td Thickening, dryness and sloughing of the epidermis

A Anterior site, exposed to the test item, 20% w/v in vehicle

P Posterior site, exposed to the test item, 10% w/v in vehicle

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance induced delayed contact hypersensitivity in 5 out of 10 (50%) guinea pigs with an intradermal induction dose of 2.5% .
Since more than 30% of the animals responded at an intradermal induction dose of more than 1%, the substance is considered as a moderate sensitizer and is therefore classified Skin Sensitizer category 1 and sub- category 1B according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.
Executive summary:

The potential of the test substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory Practice.

The induction phase was realized both by the intradermal route on day 1 (Test material 2.5% in Alembicol D (coconut oil) or vehicle) and by the cutaneous route on day 8 (Test material 60% in Alembicol D or vehicle) in 2 groups of guinea pigs: 10 in the treated group and 5 in the control group.

The challenge phase was realized on day 22 by cutaneous application of the test material at 20% and 10% in Alembicol D. The cutaneous reactions were scored 24 and 48 hours after the challenge phase.

Hexyl cinnamic aldehyde was used as a positive control and induced positive sensitization responses for nine of the ten test animals and therefore confirmed the sensitivity and reliability of the study.

In the control group, at the 24 and 48-hour readings, no cutaneous reactions were observed except in 1 animal at the 48 -hour reading that showed dryness and sloughing of the epidermis after challenge with the concentration of 20% . In the treated group, a slight erythema (grade1) was noted in 4 of the 10 animals at the 24 hour reading for both concentrations.

At the 48 hour reading, a slight or well-defined erythema (grade 1 or 2) together with a slight oedema (grade 1) was observed in 5 of the 10 animals for both concentrations. In addition, thickening, dryness and sloughing of the skin was noted in 1 animal.

The persistent cutaneous reactions observed in 5 of the 10 animals of the test item-treated group were attributed to delayed contact hypersensitivity.

It was therefore concluded that the test substance was a skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The potential of the test substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory Practice.

The induction phase was realized both by the intradermal route on day 1 (Test material 2.5% in Alembicol D (coconut oil)) and by the cutaneous route on day 8 (Test material 60% in Alembicol D) in 2 groups of guinea pigs: 5 in the control group and 10 in the treated group. The challenge phase was realized on day 22 by cutaneous application of the test material at 20% and 10% in Alembicol D. The cutaneous reactions were scored 24 and 48 hours after the challenge phase.

Hexyl cinnamic aldehyde was used as a positive control and induced positive sensitization responses for nine of the ten test animals and therefore confirmed the sensitivity and reliability of the study.

In the control group, at the 24 and 48-hour readings, no cutaneous reactions were observed except in 1 animal at the 48 -hour reading that showed dryness and sloughing of the epidermis after challenge with the concentration of 20% .In the treated group, a slight erythema (grade1) was noted in 4 of the 10 animals at the 24 hour reading for both concentrations.

At the 48 hour reading, a slight or well-defined erythema (grade 1 or 2) together with a slight oedema (grade 1) was observed in 5 of the 10 animals. In addition, thickening, dryness and sloughing of the skin was noted in 1 animal.

The persistent cutaneous reactions observed in 5 of the 10 animals of the test item-treated group were attributed to delayed contact hypersensitivity.

It was therefore concluded that the test substance was a skin sensitizer.



Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance induced delayed contact hypersensitivity in 5 out of 10 (50%) guinea pigs with an intradermal induction dose of 2.5% .

Since more than 30% of the animals responded to an intradermal induction dose of more than 1%, the substance is considered as a moderate sensitizer and is therefore classified as a Skin Sensitizer category 1 and sub- category 1B according to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.