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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:
06-Nov-2012 - 04-Feb-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
-

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isopropylnaphthalene; Ruetasolv MP
- Substance type: organic
- Physical state: colourless liquid
- Odour: aromatic, fruit-like
- Analytical purity: 96.6% (sum of isomers 1-isopropylnaphthalene, 2-isopropylnaphthalene)
- Impurities (identity and concentrations): isopropyl tetrahydronaphthalene (1.049%), diisopropyl naphthalene (1.385%)
- Isomers composition: 1-isopropylnaphthalene, 2-isopropylnaphthalene
- Lot/batch No.: 9124036031
- Expiration date of the lot/batch: 2015-03-31
- Stability under test conditions: stable in solvent
- Storage condition of test material: at room temperature (20 +/- 5°C), protected from UV light

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF
- Source: Harlan Laboratories B.V., NM Horst / The Netherlands
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: Intradermal and epidermal pretest: 326.6 - 339 g, control group and test group: 312.9 - 354.8 g
- Housing: In groups of up to ten in stainless steel cages with standard softwood bedding (‘Lignocel’)
- Diet:Teklad Global Guinea pig diet 2040C, ad libitum
- Water: ad libitum
- Acclimation period: 12 days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3°C
- Humidity (%):
- Air changes (per hr): 10 -15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
paraffin oil
Concentration / amount:
- Intradermal pretest: 50%, 25%, 10% in liquid paraffin
- Epidermal pretest: 100% (undiluted), 50%, 25%, 10% (in liquid paraffin
- Intradermal Induction (Main Test): 10% in liquid paraffin
- Epidermal Induction and challenge (Main test): 100 % (undiluted since the test item was nonirritating based on the results of the range findig pretest)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
- Intradermal pretest: 50%, 25%, 10% in liquid paraffin
- Epidermal pretest: 100% (undiluted), 50%, 25%, 10% (in liquid paraffin
- Intradermal Induction (Main Test): 10% in liquid paraffin
- Epidermal Induction and challenge (Main test): 100 % (undiluted since the test item was nonirritating based on the results of the range findig pretest)
No. of animals per dose:
- Intradermal pretest: 1
- Epidermal pretest: 2
- Control group (main test): 5
- Test group (main test): 10
Details on study design:
The intradermal induction of sensitization in the test group was performed in the nuchal region by injection of 10% of the test item in liquid paraffin and an emulsion of Freund's Complete Adjuvant and physiological saline (FCA/0.9% NaCl, 1:1).
One week after the intradermal induction, the epidermal induction of sensitization was performed by topical application of 100% of the test item for
48 hours under occlusion. The skin of the animals had been pretreated with 10% sodium laurylsulfate (SLS) approximately 24 hours prior to
application of the test item.
The animals of the control group were intradermally induced with liquid paraffin and FCA/0.9% NaCl and epidermally induced with liquid paraffin
under occlusion following pretreatment with 10% SLS.
Two weeks after epidermal induction the test and control animals were challenged by epidermal application of 100% test item on the right flank, and no vehicle was applied on the left flank.

- Injection volume (Intradermal injections): 0.1 ml/site
- Volume of formulated or undiluted test item for epidermal applications: 0.2 to 0.3 ml on a filter paper patch;
The animal's fur was shaved with a fine clipper blade just prior to treatment. The filter paper treated with test item, vehicle or 1:1 mixture of
FCA/0.9% NaCl was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and
secured with impervious adhesive tape.
The occlusive dressing was left in place for 24 hours (for the epidermal pretest and epidermal challenge) or 48 hours (for the epidermal induction).
The same patching method was used for the epidermal pretest, the epidermal induction and the epidermal challenge.

Skin reactions were evaluated at 24 and 48 hours after removal of the dressing.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde (historical data)

Results and discussion

Positive control results:
Historical data from an evaluation experiment (Harlan Laboratories Study D65975 Alpha-Hexylcinnamaldehyde)

Based on the above mentioned findings in an adjuvant sensitization test (Guinea PigMaximization test according to Magnusson & Kligman) and in accordance with Regulation (EC) No 1272/2008, alpha-Hexylcinnamaldehyde was confirmed as skin sensitizer as 70% of the test animals responded to the challenge. The sensitivity and reliability of the experimental method was confirmed (see original study: Appendix I - Positive Control).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group, not treated (left flank)
Dose level:
not treated
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical signs observerd
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group, not treated (left flank). Dose level: not treated. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no clinical signs observerd.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Control group treated with 100% test item (right flank)
Dose level:
undiluted test item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical signs observed
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Control group treated with 100% test item (right flank). Dose level: undiluted test item. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no clinical signs observed.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Test group not treated (left flank)
Dose level:
not treated
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs observerd
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Test group not treated (left flank). Dose level: not treated. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs observerd.
Reading:
1st reading
Hours after challenge:
24
Group:
other: Test group treated with 100% test item (right flank)
Dose level:
undiluted test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs observed
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: other: Test group treated with 100% test item (right flank). Dose level: undiluted test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group, not treated (left flank)
Dose level:
not treated
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical signs observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group, not treated (left flank). Dose level: not treated. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no clinical signs observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Control group treated with 100% test item (right flank)
Dose level:
undiluted test item
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no clinical signs observed
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Control group treated with 100% test item (right flank). Dose level: undiluted test item. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no clinical signs observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Test group, not treated (left flank)
Dose level:
not treated
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs observed
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Test group, not treated (left flank). Dose level: not treated. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs observed.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Test group treated with 100% test item (right flank)
Dose level:
undiluted test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs observed
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Test group treated with 100% test item (right flank). Dose level: undiluted test item. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no clinical signs observed.

Any other information on results incl. tables

Summerizing table from original report:

Skin Reactions after the Challenge

After 24 h

Positive/ Total

% Positive of Total

After 48 h

Positive/ Total

% Positive of Total

Control Group

No vehicle

(left flank)

0/5

0/5

0

0

100% test item

(right flank)

0/5

0/5

0

0

Test group

No vehicle

(left flank)

0/5

0/5

0

0

100% test item

(right flank)

0/5

0/5

0

0

Epidermal Induction: No skin reactions were observed in any animal of the test group after epidermal treatment with 100% test item following pre-treatment with 10% SLS in paraffinum perliquidum. No skin reactions were observed in the control group after treatment with the vehicle following pre-treatment with 10% SLS in paraffinum perliquidum.

Challenge: No skin reactions were observed in any of the control (previously not exposed to the test item) and test animals after the challenge with 100% test item.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the above mentioned findings in an adjuvant sensitization test (Guinea Pig Maximization test according to Magnusson & Kligman) and in
accordance with Regulation (EC) No 1272/2008, Isopropylnaphthalene; Ruetasolv MP has not to be classified as a skin sensitizer.