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Toxicological information

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
Study period:
From 16 September 1997 to 31 October 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
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:
In-vivo study already available before implementation of the LLNA method

Test material

Constituent 1
Chemical structure
Reference substance name:
Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1S,2S)-, rel-
Molecular formula:
C15H26O
IUPAC Name:
Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1S,2S)-, rel-
Constituent 2
Chemical structure
Reference substance name:
Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1R,2R)-, rel-
Molecular formula:
C15H26O
IUPAC Name:
Cyclopropanemethanol, 1-methyl-2-[[(1S,3R,5R)- 1,2,2-trimethylbicyclo[3.1.0]hex-3-yl]methyl]-, (1R,2R)-, rel-
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Javanol
- Physical state: Pale yellow viscous liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Age at delivery: 4 - 6 weeks
Age at beginning of pre-test/acclimatization period: 5-7 weeks
Body weight at pretest start: 361-369g (pretest group)
Body weight at beggining of acclimatization period: 310-431g (control and test group)
Acclimatization: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible sigs of illness were used.
Standard laboratory conditions: 10-15 air changes per hour, temeprature target at 22 +/- 3°C and relative humidity between 40-70% (values above 70% during cleaning process possible). 12 hour dark/light cycle. Recorded music was played for approximately 8 hours during the light period.
Accomodation: indivudally in Makrolon type-3 cages with standard softwood bedding.
Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos 58/97, 69/97 and 80/97 guinea pig breeding / maintenance diet, ad libitum.
Water: Community tap water from Itingen, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/l) via the drinking water was provided.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Remarks:
400
Concentration / amount:
intradermal: 5% / 0.1 ml
epicutaneous: 100% / 0.3 ml
Day(s)/duration:
intradermal at day 1, epicutaneous at day 8 (48 hours)
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
25% / 0.2 mL
Day(s)/duration:
At day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
5% / 0.2 mL (right caudal flank)
10% / 0.2 mL (right cranial flank)
Day(s)/duration:
At day 29 / 24 hours
No. of animals per dose:
15 females for main study and 3 females for pretest
Details on study design:
Pre-test
Intradermal injections: Four intradermal injections (0.1 mL/site) of 1:1 mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of 1 guinea pig. One week later intradermal injections were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1% of the test material in PEG400. The dermal reactions were assessed at 24hrs later. For intradermal induction application in the main study, a 5% test material concentation was selected.

Epidermal Applications: Four intradermal injections (0.1 mL/site) of 1:1 mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of 2 guinea pig. One week later both flanks of each guinea pig were clipped and shaved just prior to the application. Four patches of filter paper were saturated with the test article at 100, 75, 50 and 25% in PEG400 and applied to the clipped and shaved flanks. The volume of test material appleid was 0.2mL. The patches were covered by a stripof aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape to ensure intensive contact with the test material. The dressings were removed after 24hrs. Approximately 21 hours later the application site was depilated to clean the application site so that possible erythema reactions could be assessed. The reaction sites were assessed 24 and 48hrs after removal of the bandage for erythema and edema based on Draize scoring. The concentrations selected for the induction period and challenge procedure were 100 and 25%, respectively.

Main Study
Induction:
An area of dorsal skin from the scapular region was clipped free of hair. Three pairs of intradermal injections were made to the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 mixture of Freund's Complete Adjuvant and physiological saline
2) Test material diluted to 5% with PEG400
3) Test material diluted to 5% by emulsion in a 1:1 mixture of Freund's Complete Adjuvant and physiological saline
Control group:
1) 1:1 mixture of Freund's Complete Adjuvant and physiological saline
2) PEG400
3) 1:1 mixture of PEG400 in a 1:1 mixture of Freund's Complete Adjuvant and physiological saline
On test day 8 a 2x4 cm patch of filter paper saturated with undiluted test material (100%) was placed over the injection sites of the test animals. The volume of test matieral applied was approximately 0.3 m. The patch was covered with aluminum foil and secured with an elastic plaster wrapped around the trunk of the animal and secured with impervious tape to ensure intensive contact to the test material. The dressing was left in place for 48hrs. Control animals were treated as described above with PEG400 only. Reactions sites were assessed for erythema and edema 24 and 48hrs after removal of the dressing using the grading system according to Draize.

First Challenge:
Guinea pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5x5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches were saturated with the highest non-irritating concentration of 25% (left flank) or vehicle (PEG400, right flank) using the same method as for the epidermal application. The volume applied was 0.2 mL The dressing was left in place for 24 hrs. Approximately 21 hrs after removal of the dressing, the test sites treated with the test material were depilated as described in the epidermal pretest. Approximately 24 and 48hrs after removal of the dressing the application sites were assessed for eryethema and edema using the numerical scoring of Draize.

Second Challenge:
A second challenge was performed 1 week after the first challenge with the same test animals and the same test material. Treatment procedure in the second challenge was similar to the first except that two lower test material concentrations of 10 and 5% in PEG400 were used and applied to the right cranial and right caudal flank, respectively.

Challenge controls:
First Challenge:
Control guinea pigs were challenged two weeks after the epidermal induction application. Hair was clipped and shaved from a 5 x 5 cm area. Two patches were saturated with the highest non-irritating concentration of 25% (left flank) or vehicle (PEG400, right flank) using the same method as for the epidermal application. The volume applied was 0.2 mL The dressing was left in place for 24 hrs. Approximately 24 and 48 hrs after removal of the dressing the application sites were assessed for eryethema and edema using the numerical scoring of Draize.

A second challenge was performed one week after the first challenge with the same test animals. Treatment procedure in the second challenge was the same as that of the first except that two lower test article concentration of 10% and 5% in PEG 400 were used and applied to the right cranial and right caudal flank respectively.
Positive control substance(s):
yes
Remarks:
25% 2-Mercaptobenzothiazole

Results and discussion

Positive control results:
After challenge:
Control group positive/total (24hrs) positive/total (48hrs)
2-mercaptobenzothiazole 0/5 0/5
Mineral oil 0/5 0/5

Test group positive/total (24hrs) positive/total (48hrs)
2-mercaptobenzothiazole 10/10 9/10
Mineral oil 0/10 0/10

In this study 100% and 90% of the animals of the test group were observed with postive skin reaction at the 24 and 48 hour reading, respectively after treatment with a non irritant test article concentration of 25% in mineral oil. Therefore the positive control group shows a positive result for skin sensitisation after a challenge treatment with 2-mercaptobenzothiazole.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
irritation
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0% (PEG400 alone)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
irritation
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0% (PEG400 alone)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reactions observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reactions observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
4
Total no. in group:
5
Clinical observations:
erythema 1- very slight, barely perceptible
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
erythema 1- very slight, barely perceptible
Remarks on result:
positive indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reactions observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Preliminary Tests:

The degree of irritation observed after intradermal induction with 5% test material was considered suitable for intradermal treatement during the main study.

Main Study:

Epidermal application was performed and after 24 and 48hr assessments, the concentrations selected for induction period was 100% and 25% for the challenge procedure in the main study. Following the first challenge with the test material in the main study, erythema reactions of grade 1 were noted at 24 and 48hrs in the control group and therefore a second challenge was performed 1 week later using 10 and 5% of the test material. After the challenge treatment with 10 and 5% test concentrations or with vehicle alone, none of the test or control animals showed skin reactions (0/5 for controls and 0/10 for test group).

Erythema and oedema were assessed using the following numerical grading system according to Draize (Draize J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Associations of Food and Drug Officials of the United States, Austin, Texas, 1959):

ERYTHEMA AND ESCHAR FORMATION:

No erythema ............... 0

Very slight erythema (barely perceptible)......... 1

Well-defined erythema ......... 2

Moderate to severe erythema ......... 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth) ......... 4

OEDEMA FORMATION

No oedema ............. 0

Very slight oedema (barely perceptible) .......... 1

Slight oedema (edges of area well-defined by definite raising) ......... 2

Moderate oedema (raised approximately 1mm) ............. 3

Severe oedema (raised more than 1 mm and extending beyong the area of exposure) ...... 4

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Positive skin reactions were observed in the control and test group after the first challenge performed with the test article at 25% in PEG 400, considered to be of primary toxic origin (irritation).
At the second challenge no skin reactions were observed in the same control and test group treated with two lower concentrations of 10% and 5% in PEG 400.
Therefore, the test article Javanol applied at 10% and 5% in PEG 400 is considered not to be a sensitizer when used under the described test conditions.
Executive summary:

In order to assess the cutaneous allergenic potential of Javanol, the Maximization-Test in accordance with OECD Guideline No. 406 and the directive 96/54 /EEC, B.6 was carried out in 15 (10 test and 5 control) female Albino guinea pigs. The study was designed and performed according to Good Laboratory Practice standards.

The intradermal induction of sensitization was carried out with a 5% dilution of the test article in polyethylene glycol (PEG400) and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitization was conducted under occlusion with the undiluted test article (100%). Two weeks after the epidermal induction application the first challenge was completed by epidermal application of the test article at 25% in PEG 400 under occlusive dressing. The animals of the control group were induced with PEG400 and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal the dressing.

Erythematous reactions after the first challenge procedure:

 positive / total after 24 hours  positive / total after 48 hours
 Control group - Javanol 25%  4/5  5/5
 Control group - PEG 400 only  0/5  0/5
 Test group - Javanol 25%  7/10  7/10
 Test group - PEG 400 only  0/10

 0/10

As positive skin reactions were observed in the control group after the first challenge, a second challenge was performed in the control and test group one week after the first challenge using two lower test article concentrations of 10 and 5%.

Erythematous reactions after the second challenge procedure:

 positive / total after 24 hours  positive / total after 48 hours
 Control group - Javanol 10%  0/5  0/5
 Control group - Javanol 5%  0/5  0/5
 Test group - Javanol 10%  0/10  0/10
 Test group - Javanol 5%  0/10

 0/10

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

No deaths occured.

Therefore, the test article Javanol applied at 10% and 5% in PEG 400 is considered not to be a sensitizer when used under the described test conditions.

Concluding from these results and applying the CLP and GHS criteria, the test substance should not be classified as sensitizing to the skin according to the Regulation (EC) N° 1272 -2008 (CLP) and the GHS.