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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:
06-09 March 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
yes
Remarks:
Since no signs of irritation were observed to the highest test substance concentration epidermally tested in the preliminary irritation study, the test site of all animals should have been treated 10% SDS approximately 24 hours before the epidermal induct
Qualifier:
according to guideline
Guideline:
other: European Community (EC), Council Directive 67/548/EEC, Annex V, Part B, Methods for the Determination of Toxicity, as last amended by Commission Directive 96/54/EC, Annex IV C, B.6: "Skin sensitisation",
Version / remarks:
1996
Qualifier:
according to guideline
Guideline:
other: Allergic Contact Dermatitis in the Guinea-Pig: Identification of Contact Allergens" Magnusson B. Kligman A.M., 1970 published by C.C. Thomas, Springfield, Illinois, USA.
Version / remarks:
1970
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Maximisation test is selected because it is regarded as the most sensitive and the preferred method with regard to testing for sensitisation potential at the time of the performance of this study.

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
439-270-3
EC Name:
-
Cas Number:
260408-02-4
Molecular formula:
CAS formula: (C12 H10 O4 S . C6 H6 O . Cl5 P . Cl H4 N)x Molecular formula of the reaction products: (C12 H10 N O2 P)n (n=3-15)
IUPAC Name:
439-270-3
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): SPS-100
- Physical state: White to pale yellow powder
- Storage condition of test material: In refrigerator (0-14°C) in the dark
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 Deutschland, Kisslegg, Germany.
- Age at study initation: Young adult animals (approx. 7 weeks old)
- Females nulliparous and non-pregnant: Yes
- Weight at study initation: 405 - 521g
- Housing: Group housing of 5 animals per labelled metal cage with wire-mesh floors.
- Diet: Free access to standard guinea pig diet, including ascorbic acid (1000 mg/kg); (Charles River Breeding and Maintenance Diet for Guinea Pigs, Altromin, Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 20 - 70
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
2% for the intradermal induction and 50% for the epidermal induction.
Day(s)/duration:
Intradermal induction: 7 days. Epidermal induction: 48 hours.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50% for the challenge phase.
Day(s)/duration:
24 hours
No. of animals per dose:
Test animals: 10
Control animals: 5
Details on study design:
RANGEFINDING TESTS:
Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals selected were between 4 and 9 weeks old. No body weights were determined at termination.

Intradermal injections:
A series of four test substance concentrations was used, the highest concentration being 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 injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. 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.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Site: The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area as follows:
1) 0.1 mL: FCA (50% in water for injection)
2) 0.1 mL: test substance at 2% concentration (control animals: 0.1 mL corn oil)
3) 0.1 mL: 1:1 mixture of the test substance at a 4% concentration + FCA (undiluted)
One of each pair was on each side of the midline and from cranial 1) to caudal 3).
- 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) 50% 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 50% test substance and the vehicle)
- Day of challenge: day 22
- Exposure period: 24 hours (occlusive)
- Site: flank
- Amount: 0.1 mL
- Readings: scores were rated 24 and 48 hours after patch removal

OBSERVATIONS
Mortality/Moribundity checks: twice daily
Toxicity: At least once daily.
Body weights: Animals were weighed individually on Day 1 (pre-dose) and at termination of the study
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.
Challenge controls:
Not applicable.
Positive control substance(s):
yes
Remarks:
the results of a reliability check, performed in January/February 2001 with 10% Alpha-Hexylcinnamaldehyde, are reported.

Results and discussion

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:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% Alpha-Hexylcinnamaldehyde
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:
10% Alpha-Hexylcinnamaldehyde
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Maximum concentration not causing irritating effects in preliminary test: 50 %

Signs of irritation during induction:
Erythema grade 1 (2/10), 2 (6/10) or 3 (2/10).

Evidence of sensitisation of each challenge concentration:
0/0

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 maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and in accordance with GLP principles, showing a sensitization rate of 0 per cent.
Executive summary:

In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and in accordance with GLP principles. Ten guinea pigs were intradermally injected with a 2% concentration and epidermally exposed to a 50% concentration of the test substance. Five control animals were similarly treated, but with vehicle alone (corn oil). Slight to moderate erythema was observed during the intradermal induction (conc. 2%) at the injection sites in the test animals. Slight erythema was observed following the epidermal induction (conc. 50%) in all test animals. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. No skin reactions were evident after the challenge exposure in the experimental and control animals. Based on these results the substance 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 items and mixtures (including all amendments).