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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:
11 Aug 2003 - 02 Oct 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1992)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Arbeitsschutz, Arbeitsmedizin und Sicherheitstechnik, München, Germany
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The current accepted and preferred method for skin sensitisation testing according to the REACH legislation (EC no 1907/2006) and CLP Regulation (EC No 1272/2008) is the murine local lymph node assay (LLNA). A validated test method, OECD TG 429 (OECD 2002) is available for the LLNA. The guideline acknowledges the limits of the LLNA, and states that there are instances where test substance classes or substances containing functional groups shown to act as potential confounders make the use of guinea pig tests more appropriate. It is concluded that the LLNA is not applicable where the properties of the test material cause interference in the accuracy of the LLNA (OECD 2002). The statement in the OECD TG 429 is given with reference to the findings of Basketter et al. (2009a), who demonstrated false positives in silicone based substances which had previously been demonstrated to be non-sensitisers in the guinea pig maximisation test (GPMT). The importance of available evidence from guinea pig results, consideration of chemical reactivity, epidermal bioavailability and clinical and experimental human data are emphasised as central to reaching appropriate regulatory decisions for substances which have been shown to fall outside the specificity of the LLNA (Basketter et al., 2009b). The non-applicability of the LLNA for silicone based substances has also been demonstrated by Petry et al. (2012). The sensitisation potential of polyfunctional silicone materials was tested in a comparative study investigating the GPMT and the LLNA assays, which found the five tested substances to be negative in the GPMT whereas they were concluded to be weak to moderate skin sensitisers in the LLNA (Petry et al., 2012).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Silane 449007 VP
- Physical state: liquid
- Storage condition of test material: 5±3°C, protected from light

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: 300-500 g
- Fasting period before study: no
- Housing: animals were barrier maintained (semi-barrier) in an air-conditioned room, animals were kept in groups in Terluran-cages on Altromin saw fiber bedding
- Diet: Altromin 3122 maintenance diet for guinea pigs (totally pathogen-free, rich in crude fiber), ad libitum
- Water: tap water, ad libitum
- Acclimation period: adequate acclimatisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
10%
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
2
Challenge
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
1
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Range-finding assay: 4
Main test: 5 controls and 10 test animals
Details on study design:
RANGE FINDING TESTS:
2 animals were intradermally treated with 1%, 5%, 10%, 25%, 50% of the test item (diluted in corn oil). For the 50% concentration erythema grade 3 and necrosis (0.5 cm diameter) were observed at both 24 and 48 h post-exposure. For the 25% concentration erythema grade 2 and necrosis (0.3 cm diameter) were observed at both 24 and 48 h post exposure. For the 10% concentration erythema grade 1 was observed 24 h only. For 5% and 1% no signs of irritation or toxicity were observed. Therefore the concentration of 10% was chosen for the intradermal induction.
2 further animals were topically treated with 50% and 100% concentration for 24 and 48 h, respectively. No signs of irritation and systemic toxicity were recorded during the observation time for the animals. Therefore the 100% concentration was chosen for the topical induction as well as for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/isotonic saline
Injection 2: test substance in corn oil
Injection 3: test substance in a 1:1 mixture (v/v) FCA/isotonic saline

Epicutaneous: test substance (undiluted)

- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/isotonic saline
Injection 2: Corn oil
Injection 3: Corn oil at 50% (w/v) in a 1:1 mixture (v/v) FCA/isotonic saline

Epicutaneous: Corn oil

- Site: shoulder region (intradermal + epicutaneous)
- Frequency of applications: every 7 days
- Duration: Days 0-9
- Concentrations:
Test item: intradermal 10%, epicutaneous 100%
Control: intradermal 0%, epicutaneous corn oil


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: day 20
- Exposure period: 24 h
- Test groups: 0.5 ml of the undiluted test item
- Control group: 0.5 ml of the undiluted test item
- Site: left flank (test item), right flank (vehicle)
- Concentrations: 100%
- Evaluation (hr after challenge): 24, 48, and 72
Challenge controls:
The control group is actually a challenge control
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole

Results and discussion

Positive control results:
The positive control substance (15% mercaptobenzothiazole in Vaseline) induced positive reactions in 8/10 animals (80%), thus meeting the reliability criteria for the GPMT test (≥ 15% positive response). Positive control substances are used periodically as a reliability check (last check before study May 2013) and not during the study itself.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Induction: 0%; challenge: 100%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Induction: 0%; challenge: 100%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Induction: 10%; challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Induction: 10%; challenge: 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Induction: 2%; challenge: 15%
No. with + reactions:
8
Total no. in group:
10

Any other information on results incl. tables

Induction readings: 7 animals showed erythema grade 1 at 24 h (induction first stage). No signs of irritation were observed after the topical application (induction second stage). No signs of irritation and no signs of general toxicity were observed after challenge. Body weights were comparable within the test and control groups and the historical controls.

Applicant's summary and conclusion

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
In a GPMT according to OECD guideline 406 and in compliance with GLP, the test item was not sensitising. Induction (intradermal: 10%; topical: 100%) and challenge (100%) with the test item revealed no skin reactions in any of the 10 animals. Furthermore no skin reactions were observed in the negative controls (induction with corn oil, topical challenge with 100% test item).