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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:
13 August, 2012 - 09 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions.
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Previously, a LLNA study was performed. However, for a NICNAS notification in Australia further data was required, as the available information was not considered sufficient to reverse the classification that was based on the LLNA study. Therefore a GPMT study was conducted.

Test material

Constituent 1
Test material form:
liquid
Details on test material:
- Name of test material: Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol
- Physical state: Light brown liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany.
- Age at study initiation: Young adult animals (approx. 4 weeks old)
- Weight at study initiation: 239 - 306 g
- Housing: Group housing of maximally 5 animals per labeled Noryl cage.
- Diet: Complete maintenance diet for guinea pigs (MS-H ered, SSNIFF® Spezialdiäten, GmbH, Soest, Germany). In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
20% for the intradermal induction and 100% for the epidermal induction.
50% for the challenge phase.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
20% for the intradermal induction and 100% for the epidermal induction.
50% for the challenge phase.
No. of animals per dose:
Test animals: 20
Control animals: 10
Details on study design:
RANGE FINDING TESTS: (4 animals, age: between 4 and 9 weeks old)

Intradermal injections:
A series of four test substance concentrations was used (10, 20, 50 and 100%). Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
- Result: A 20% test substance concentration was selected for the main study: necrosis was observed at 100 and 50%, while only erythema was observed at 20% (grade 2 at 24h, grade 1 at 48h).

Epidermal application:
A series of four test substance concentrations was used (10, 20, 50 and 100%) Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.
- Result: A 100% concentration was selected for the epidermal induction exposure as no signs of necrosis were present but only slight irritation was observed.

MAIN STUDY
The Main study was performed in a step wise fashion. Initially, ten experimental and five control animals were treated. Based on the results, the study was extended with another set of ten experimental and five control animals.
A. INDUCTION EXPOSURE
- No. of exposures: 1

1) Intradermal injections on day 1:
- Site: scapular region. One of each pair was on each side of the midline and from cranial to caudal:
Three pairs of intradermal injections:
1) 0.1 mL: FCA (50% in water for injection)
2) 0.1 mL: test substance at a 20% concentration (control animals: 0.1 mL corn oil)
3) 0.1 mL: 1:1 mixture of the test substance at a 40% concentration + FCA (undiluted)
- Readings: on day 3 (48 hrs after the injections)

2) Topical application on day 8:
- Amount: 0.5 mL (control animals: 0.5 mL corn oil) 100% test substance
- Area: approximately 6 cm^2
- Exposure period: 48 hours (occlusive)
- Readings: scores were rated directly after patch removal

B. CHALLENGE EXPOSURE (all animals, with the 50% test substance and the vehicle)
- Day of challenge: day 21 or 22
- Exposure period: 24 hours (occlusive)
- Site: flank
- Amount: 0.1 mL
- Readings: scores were rated 24 and 48 hours after patch removal

RECHALLENGE: Day 30, like first challenge, in first set of animals.

OBSERVATIONS
Mortality/Viability: Twice daily
Toxicity: At least once daily.
Body weights: Prior to start and after (each) challenge.
Necropsy: No necropsy was performed.
Irritation: Skin reactions were graded according to OECD 406. The intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis. A description of all other local effects was recorded.
To facilitate scoring, the epidermally treated skin-areas were clipped at least 3 hours before the next reading.
Challenge controls:
Not applicable.
Positive control substance(s):
yes
Remarks:
the results of the latest reliability check, performed in May/June 2012 with Alpha- Hexylcinnamaldehyde, are reported.

Results and discussion

Positive control results:
The latest reliability check shows a sensitisation rate of 80%

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 20.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
(first set of animals were rechallenged)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: (first set of animals were rechallenged).
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
(first set of animals were rechallenged)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: (first set of animals were rechallenged).
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
(first set of animals were rechallenged)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: (first set of animals were rechallenged).
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
(first set of animals were rechallenged)
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: (first set of animals were rechallenged).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
other: Not classified.
Remarks:
According to Regulation (EC) 1272/2008
Conclusions:
In a guinea pig maximisation test method the potential of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol for skin sensitisation was tested according to OECD 406 guideline and GLP principles, showing a sensitization rate of 5 per cent.
Based on these results Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.
Executive summary:

In a guinea pig maximisation test method the potential of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol for skin sensitisation was tested according to OECD 406 guideline and GLP principles.

Slight to moderate erythema was observed during the intradermal induction (conc. 20%) at all injection sites in control and test animals.

Slight erythema was observed following the epidermal induction (conc. 100%) in 12 test animals and 3 control animals.

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

In the first set of ten experimental and five control animals, the first challenge resulted in skin reactions of discrete or patchy erythema in one experimental animal in response to the 50% test substance concentration. No skin reactions were evident in the control animals. To confirm the results of the first challenge, the first challenge was repeated in a second challenge one week later. The second challenge results were the same as for the first challenge.

Based on the test guidelines, a second set of ten experimental and five control animals were treated in the same way as the first set. No skin reactions were evident after the challenge exposure in this second set of animals. These results indicate a sensitization rate of 5 per cent.

Based on these results Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.