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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:
weight of evidence
Study period:
From November 08, 2011 to December 27, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to standard guidelines in compliance with GLP; adequate consistency between data, comments and conclusions.
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA test is available and was included to avoid unnecessary testing according to the Regulation (EC) No 1907/2006.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: 12-hydroxystearic acid
- Physical state: pale yellow solid (flakes)
- Lot/batch No.: 10B2122
- Expiry date: April 2012
- Storage condition: at room temperature.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: at the beginning of the treatment period, the animals of the main test were 5 weeks old
- Mean body weight at study initiation: the males had a mean body weight of 326 g (range: 298 g to 351 g) and the females a mean body weight of 308 g (range: 282 g to 328 g)
- Fasting period before study: no
- Housing: polycarbonate cages with stainless steel lid
- Diet: 106 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00).

IN-LIFE DATES: 08 November 2011 to 27 December 2011.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Concentration / amount:
Induction phase:
Concentration for intradermal injection: 2.5% *
Concentration for topical application: 10% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 2.5%**
** highest non-irritant concentration based on preliminary assays
Challengeopen allclose all
Route:
epicutaneous, occlusive
Concentration / amount:
Induction phase:
Concentration for intradermal injection: 2.5% *
Concentration for topical application: 10% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application: 2.5%**
** highest non-irritant concentration based on preliminary assays
No. of animals per dose:
- preliminary test: 6 treated animals
- main test: 30 treated animals
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: topical induction: 48 h
- Site: interscapular region
- Frequency of applications: once intradermal, once topical.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 4 weeks between injection (induction) and challenge
- Exposure period: 24h
- Site: right flank (test item) and left flank (vehicle)
- Evaluation (hr after challenge): 24, 48 and 72 h after removal of dressing.
Positive control substance(s):
not required
Remarks:
mercaptobenzothiazole tested in another study

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
discrete-moderate erythema
Remarks on result:
other: Reading: 1st reading. Hours after challenge: 24.0. Group: negative control. Dose level: 2.5%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: discrete-moderate erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5%
No. with + reactions:
6
Total no. in group:
10
Clinical observations:
discrete-moderate erythema
Remarks on result:
other: Reading: 2nd reading. Hours after challenge: 48.0. Group: negative control. Dose level: 2.5%. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: discrete-moderate erythema.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2.5%
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
discrete-moderate erythema
Remarks on result:
other: Reading: 1st reading. Hours after challenge: 24.0. Group: test group. Dose level: 2.5%. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: discrete-moderate erythema.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2.5%
No. with + reactions:
9
Total no. in group:
20
Clinical observations:
discrete-moderate erythema
Remarks on result:
other: Reading: 2nd reading. Hours after challenge: 48.0. Group: test group. Dose level: 2.5%. No with. + reactions: 9.0. Total no. in groups: 20.0. Clinical observations: discrete-moderate erythema.

Applicant's summary and conclusion

Interpretation of results:
other: doubtful
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The potential of the test substance to induce delayed contact hypersensitivity in guinea pigs remained doubtful. In order to conclude, a further study should be carried out using a lower concentration for the challenge application.
Executive summary:

The objective of this study was to evaluate the potential of the test substance, 12-hydroxystearic acid, to induce delayed contact hypersensitivity in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD Guideline 406 (17th July 1992) and Commission Regulation (EC) (No. 440/2008, B.6, 30 May 2008). This study was conducted in compliance with the principles of Good Laboratory Practice.

 

A preliminary test was first performed in order to determine the test item concentrations to be used in the main test.

In the main test, 30 guinea pigs were allocated to 2 groups: a control group of 5 males and 5 females and a treated group of 10 males and 10 females.

 

On Day 1, three pairs of intradermal injections were performed in the interscapular region of animals:

.          Freund's Complete Adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl,

.          test item at the concentration of 2.5% in corn oil (treated group) or vehicle alone (control group),

.          test item at the concentration of 2.5% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in FCA/0.9% NaCl (50/50, v/v) (control group).

 

As in the preliminary test, the highest well-tolerated concentration was shown to be slightly irritant after topical application, 0.5 mL of sodium lauryl sulfate at 10% (w/w) in vaseline was applied to the induction site on Day 7 in order to induce a local irritation.

 

On Day 8, a filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 10% (w/w) in corn oil, and then applied to the clipped interscapular region, over the intradermal injection sites. The filter paper was held in place by means of an occlusive dressing for 48 h. The presence of local irritation was checked (but not scored). The control group animals received an application of the vehicle under the same experimental conditions.

 

The induction phase was followed by a 14 day rest period.

 

On Day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 2.5% (w/w) in corn oil to the right flank. The chamber was held in contact with the skin by an occlusive dressing for 24 h. The vehicle was applied to the left flank under the same experimental conditions.

 

Cutaneous reactions were evaluated before treatment and 24, 48 and 72 h after removal of the dressing.

 

Each animal was observed at least once a day for mortality and clinical signs during the treatment and observation periods. Bodyweight was recorded on Day 1 and at the end of each observation period.

Upon completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination. For all animals, skin samples of the challenged application sites were preserved. No microscopic examination was performed.


Results can be summarised as follows:

 

Mortality, clinical signs and body weight

No unscheduled deaths occurred during the main test.

No clinical signs indicative of systemic toxicity were observed in any animals.

The bodyweight change of the treated animals was similar to that of controls.

 

Cutaneous reactions

At the 24 h reading, after the challenge application, no cutaneous reaction was observed on the left flank of control animals (vehicle application). Discrete or moderate erythema were noted on the right flank (test item-treated) of 5/10 control animals.

In the test item-treated group, at the 24 h reading, a discrete erythema was noted on the left flank (vehicle treated) of one male only. A discrete or moderate erythema was noted on the right flank (test item‑treated) of 7/20 animals.

 

At the 48 h reading, a discrete or moderate erythema was observed on the left flank of 4/10 control animals (vehicle application). Discrete or moderate erythema were noted on the right flank (test substance‑treated) of 6/10 control animals.

In the test substance-treated group, at the 48 h reading, a discrete erythema was noted on the left flank (vehicle treated) of 6/20 animals. A discrete or moderate erythema was noted on the right flank (test substance‑treated) of 9/20 animals.

The increase in the incidence and severity of cutaneous reactions on both flanks was correlated with the shaving of the flanks after the 24 h reading.

 

At the 72 h reading, a discrete erythema was observed on the left flank (vehicle application) of only three males of the control group. Discrete or moderate erythema was noted on the right flank (test substance‑treated) of the same males.

In the test substance-treated group, at the 72 h reading, a discrete or moderate erythema was noted on the left flank (vehicle treated) of 5/20 animals. A discrete erythema sometimes associated with dryness of the skin was noted on the right flank (test substance-treated) of 6/20 animals.

 

At each observation time, dryness of the skin was observed in both group, associated with erythema or not.

 

It was not possible to determine the incidence of sensitisation responses in the induced test substance-treated group as:

.            after the 24 h observation, the shaving of the flanks elicited cutaneous reactions on the left flank (vehicle treated) of all animals,

.            the skin reactions seen on the right flank (test substance-treated) of the control group animals were indicative of skin irritation since none of these animals were exposed to the test substance prior to the topical challenge.

The concentration of the test substance selected for the topical challenge was too high.

Following these observations, it was therefore not possible to give a clear conclusion on this study.

Conclusion

The potential of the test substance to induce delayed contact hypersensitivity in guinea pigs remained doubtful.

In order to conclude on the dermal sensitization potential of the test substance, a further study should be carried out using a lower concentration for the challenge application.