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Diss Factsheets

Administrative data

Description of key information

GPMT according to OECD TG 406 and in compliance with GLP regulations: sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(1981)
Deviations:
yes
Remarks:
Sensitivity check by using a positive control substance was performed only once a year.
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(1984)
Deviations:
no
GLP compliance:
yes
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).
Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 339-476 g
- Housing: The animals were housed under standard laboratory conditions. The animals were housed in groups of two animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The animal was identified by eartags.
- Diet (ad libitum): standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4 mm (Hope Farms, Woerden, The Netherlands). In addition, hay (Broekman Institute, Someren, The Netherlands) was provided once a week. Certificates of analysis were examined and then retained in the RCC NOTOX archives.
- Water (ad libitum): tap-water diluted with decalcified water. Certificates of analysis were examined and then retained in the RCC NOTOX archives.
- Acclimation period: at least 5 days before start of treatment under test conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 (fluctuations were observed, but were not considered to be relevant)
- Humidity (%): 55 (fluctuations were observed, but were not considered to be relevant)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
5%
Route:
other: epicutanoeus
Concentration / amount:
50%
Route:
epicutaneous, semiocclusive
Vehicle:
paraffin oil
Concentration / amount:
5, 10, 25%
No. of animals per dose:
- 20 females (experimental group)
- 10 females (control group)
Details on study design:
RANGE FINDING TESTS:
A preliminary study was carried out to investigate suitable irritant test substance concentrations for the induction phase and suitable non-irritant test substance concentrations for the challenge.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 hours (epicutaneous)
- Test groups: Intradermal (3 pairs of injections):
Injection 1: the test substance diluted to 5% (w/w) with paraffin oil
Injection 2: Freund's Complete Adjuvant (FCA, Difco, Detroit, USA), 50:50 with distilled water for injection (pyrogen free, Fresenius AG, Bad Homburg, Germany)
Injection 3: the test substance, at twice the concentration used in injection 1, emulsified in a 50:50 mixture of Freund's Complete Adjuvant
Epicutaneous: 0.5 ml of the test substance (50% w/w) in paraffin oil
- Control groups: treated and assessed as described for the test groups with the omission of the test substance
- Site: an area of 4 x 6 cm of the dorsal skin from the scapular region
- Frequency of applications: every 7 days
- Duration: Day 1-8
- Concentrations: intradermal 5%, epicutaneous 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: Day 22 (two weeks after epidermal induction application)
- Exposure period: 24 h
- Test groups: test substance and vehicle only (0.05 ml each)
- Site: left flank
- Concentrations: 25, 10, and 5% test substance in vehicle
- Evaluation (hr after challenge): 24 and 48 h

OTHER: In addition to the skin reactions the following data were recorded:
- Mortality/viability/toxicity: once daily
- Body weights: prior to start and at termination of the study
Challenge controls:
The control group is actually a challenge control.
Positive control substance(s):
yes
Remarks:
(historical, most recent test carried out in June 1992 by using formaldehyde)
Positive control results:
Clearly positive results were observed after treatment of Himalayan guinea pigs with the positive control substance formaldehyde, concluding that the animals were an appropriate model. One restriction was observed, as the sensitivity was only observed once a year, and not every 6 months as stated in the OECD TG 406 (adopted 1992).
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
One animal showed discolouration as a sign of necrosis and one animal showed scaliness.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
none
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%, 5%, and vehicle only
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
4 animals showed discolouration as a sign of necrosis and 16 animals showed scaliness.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
2 animals showed discolouration as a sign of necrosis and 13 animals showed scaliness.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
11
Total no. in group:
20
Clinical observations:
4 animals showed scaliness.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%, 10%, 5%, and vehicle only
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Group:
positive control
Remarks on result:
positive indication of skin sensitisation

PRELIMINARY STUDY:

No signs of systemic toxicity werre observed during the preliminary study. However, body weight loss was noted in one of the five animals.

 

MAIN STUDY:

Induction: None of the experimental or control animals showed erythema or edema after the 48 h occluded epidermal induction exposure.

Challenge: 24 h after challenge exposure 5 animals of the control group showed a skin reaction in response to the 25% test substance concentration only. No skin reactions were noted in response to the other concentrations. In contrast, 20, 19, and 10 animals showed a skin reaction in response to the 25, 10, and 5% test substance concentrations, respectively. These reactions were characterised by redness, signs of necrosis, swelling, and scaliness.

 

No symptoms of systemic toxicity were observed in the animals of the main study during the study period. No mortality occurred during this main study. The average body weight gain of experimental and control animals was similar.

Interpretation of results:
other: CLP/EU GHS Category 1B (H317) according to Regulation (EC) No 1272/2008
Conclusions:
The test item was tested for skin sensitization according to the OECD TG 406 (1987, guinea pig maximisation test) and in compliance with GLP. No mortality occured and no signs of systemic toxicity were observed. After challenge exposure 20, 19 and 10 animals in the test group showed a skin reaction at the first reading in response to 25, 10, and 5% test substance concentration, respectively. Whereas only 5 animals of the control group challenged with 25% substance concentration showed skin reactions. Hence, the test item has to be classified as a sensitizer (Cat 1B) according to EC/1272/2008.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Studies were chosen as key when the available study was of relevance and of sufficient quality for classification, labelling and for risk assessment.

In a key GPMT, conducted in compliance with GLP and according to OECD TG 406, the test substance was found to be a skin sensitiser. Induction was performed twice (day 1: intradermal; day 8: epicutaneous) with 5% (intradermal) and 50% (epicutaneous, 0.5 ml, 48 h exposure) test substance (NOTOX, 1993g). Two weeks after dermal induction application a challenge was conducted, where 0.05 ml of a 5, 10, and 25% test substance solution was applied. Readings were performed 24 and 48 h after the start of the challenge treatment.

None of the experimental or control animals showed erythema or oedema after the 48 h occluded epidermal induction exposure. At challenge, 5 animals of the control group showed a skin reaction in response to the 25% test substance concentration. No skin reactions were noted in response to the other concentrations. In contrast, twenty, nineteen, and ten animals showed a skin reaction in response to the 25, 10, and 5% test substance concentrations, respectively. These reactions were characterised by redness, signs of necrosis, swelling, and scaliness.

No symptoms of systemic toxicity were observed in the animals of the main study during the study period. No mortality occurred during this main study. The average body weight gain of experimental and control animals was similar.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Due to more than 30% positive responders in the GPMT after intradermal induction with 5% test substance solution, the test item has to be classified as a sensitizer (Cat 1B, H317) according to EC/1272/2008.