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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:
10 June 2016 - 28 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Guinea-Pig Miximization Test (GMPT), 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF) Guidelines
Version / remarks:
2000, including the most recent revisions
Deviations:
no
Principles of method if other than guideline:
The study was based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" (1970).
GLP compliance:
yes (incl. certificate)
Remarks:
certificate dated 3 November 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Maximization test was selected since the test substance is an amino sugar, structurally similar to glucosamine. Glucosamines are naturally occurring substance that are known to be involved in the regulation of T-lymphocytes. Due to the structural similarity to glucosamines and thus the possibility of immunomodulating effect, the local lymph node assay (LLNA) is not considered to be the appropriate test system. The test substance may induce unspecific polyclonal suppressing/ compensatory proliferating effect on the lymphocytes, which would lead to increased SI values. In such case, a false positive response would be obtained in the LLNA.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Specific details on test material used for the study:
Solubility in vehicle:
• Water: >583 g/L
Stability in vehicle:
• Water: Stable

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
- Source: Charles River France, L’arbresle, France.
- Age at study initiation: Young adult animals (approx. 4 weeks old)
- Weight at study initiation: 257 - 303 g
- Housing: Animals were group housed in labeled Noryl cages containing sterilized sawdust as bedding material and shelters as cage enrichment.
- Diet: Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany). In addition, hay was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 June 2016 to 28 July 2016

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
water
Concentration / amount:
0.5% test item, 1:1 w/w mixture of the test item (1%) and Freunds' Complete Adjuvant and 1:1 w/w mixture of water and Freunds' Complete Adjuvant (all 0.1 mL/site)
Day(s)/duration:
1
Adequacy of induction:
other: the highest concentration that did not induce local necrosis
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
50% test item concentration (0.5 mL)
Day(s)/duration:
9 (48 hrs)
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
50% test item concentration (0.1 mL)
Day(s)/duration:
22
Adequacy of challenge:
other: maximum concentration that could technically be applied
No. of animals per dose:
5 females (control), 10 females ( experimental group)
Details on study design:
RANGE FINDING TESTS:
The test system and procedures were identical to those used during the main study
Intradermal injections:
- concentrations tested: 50%, 20%, 10%, 5%. The highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hrs after treatment.
Based on the results in the initially treated animals, two additional animals were treated in a similar manner with 2%, 1%, 0.5% and 0.2% at a later stage.

Epidermal application:
concentrations tested: 50%, 20%, 10%, 5%.; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied per animal.

Based on the results, the test item concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure.
No signs of irritation were observed to the highest test item concentration epidermally tested. Therefore, the test site of all animals of the main study was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction.
A 50% test item concentration was selected for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
- Number of exposures: two, one intradermal and one epidermal

intradermal injection:
- Exposure period: -
- Test groups: water+FCA, test item, test item + FCA
- Control group: water+FCA, vehicle, vehicle+FCA
- Site: scapular region
- Frequency of applications: once, onday 1
- Concentrations: 0.5% test item concentration (0.1 mL /site),
- Reading dermal reactions: Day 3

epidermal:
- Skin was pre-treated with 10% SDS on day 8
- Exposure period: 48 hrs using Metalline Patch after which skin was cleaned of residual test item
- Test groups: test item
- Control group: vehicle
- Site: scapular area between the injection sites
- Frequency of applications: day 9
- Concentrations: 50% test item concentration (0.5 mL /site),
- Reading dermal reactions: 48 hrs after epidermal application

B. CHALLENGE EXPOSURE
- Number of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hrs using Patch Test Plasters, after which skin was cleaned of residual test item
- Test groups: test item
- Control group: test item
- Site: flank
- Concentrations: 50% test item concentration (0.1 mL/site)
- Evaluation (hr after challenge): 24 and 48 hrs
Challenge controls:
test item concentration 50%
Positive control substance(s):
yes
Remarks:
a reliability check with Alpha-hexylcinnamaldehyde is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques

Results and discussion

Positive control results:
The reliability check with Alpha-hexylcinnamicaldehyde indicates that the GMPT as performed at Charles River Laboratories Den Bosch is an appropriate model for the evaluation of the sensitizing potential of a test item in a Maximization type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% test item concentration
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% test item concentration
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% test item concentration
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% test item concentration
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Preliminary Irritation Study

Based on the results, the test item concentrations selected for the main study were a 0.5% concentration for the intradermal induction and a 50% concentration for the epidermal induction exposure. No signs of irritation were observed to the highest test item concentration epidermally tested. Therefore, the test site of all animals of the main study was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction. A 50% test item concentration was selected for the challenge phase.

Main study

FCA = Freunds' Complete Adjuvant

Grading erythema: 0 = No erythema 1 = Slight erythema (barely perceptible) 2 = Well-defined erythema 3 = Moderate erythema

Induction Phase

Erythema readings day 3 (intradermal exposure)

Controls

1:1 mixture of FCA and water: score 2 (2/5), 3 (3/5)

Vehicle: score 0 (5/5)

1:1 mixture FCA and vehicle: score 2 (3/5), 3 (2/5)

Test group

1:1 mixture of FCA and water: score 2 (1/10), 3 (9/10)

0.5% test item : score 1 (9/10), 2 (1/10)

1:1 mixture FCA and 1% test item: score 3 (1/10) other 9 animals showed signs of necrosis at injection site

Erythema, oedema readings day 10 (epidermal exposure)

Controls

Vehicle: erythema: score 0 (2/5), 1 (2/5), 2 (1/5), no oedema was observed in any of the animals

Test group

50% test item: erythema: score 1 (1/10), 2 (8/10), 3 (1/10), no oedema was observed in any of the animals

The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

Challenge Phase

No skin reactions were evident after the challenge exposure in the experimental and control animals.

Toxicity / Mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In a Guinea-Pig Maximization Test, performed according to OECD/EC test guidelines and in compliance with GLP, DMEGA-100 was considered not to be a skin sensitiser, as the sensitization rate was 0%.
Executive summary:

Contact hypersensitivity to DMEGA-100 was assessed in the Albino Guinea Pig (Maximisation-Test) according to OECD/EC test guidelines and in compliance with GLP principles. Test item concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with 0.5% DMEGA-100 on day 1 (highest concentration that did not induce local necrosis in the preliminary study) and epidermally exposed to a 50% concentration of DMEGA-100 on day 9. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. On day 22, all animals were epidermally challenged with a 50% test item concentration (maximum concentration that could technically be applied) and the vehicle. Adequate negative and positive control groups (alpha-hexylcinnamicaldehyde) were included.

Intradermal injection with DMEGA-100 resulted in slight erythema on day 3 and epidermal exposure resulted in well-defined erythema. No skin reactions were evident after the challenge exposure in the experimental and control animals. There was no evidence that DMEGA-100 had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 50% test item concentration in the challenge phase. This result indicates a sensitization rate of 0%. The reliability check with Alpha-hexylcinnamicaldehyde indicates that the GMPT is an adequate test system for the evaluation of the sensitizing potential of a test item.

Based on these results DMEGA-100 does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and EC criteria for classification and labelling requirements for dangerous items and preparations (Council Directive 67/548/EEC) (including all amendments).