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Key value for chemical safety assessment

Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 24, 2001 - May 9, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(draft of November 2000)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Name of test material (as cited in study report): CITRATHAL R
- Description: Colourless to pale yellow liquid
- Analytical purity: not applicable (as it is an UVCB substance)
- Composition of test material, percentage of components: as defined in section 1.2
- Lot/batch No.: 9000416199
- Expiration date of the lot/batch: August 27, 2001
- Storage condition of test material: In the original container, at room temperature (range of 17-20 'C), away from direct sunlight
Species:
mouse
Strain:
other: CBA/JIbm
Sex:
female
Details on test animals and environmental conditions:
- Source: RCC Ltd., Biotechnology & Animal Breeding Division, Wölferstrasse 4, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 7-12 weeks (beginning of acclimatization)
- Weight at study initiation: 18.5-22.4 g (beginning of acclimatization)
- Housing: Animals were group housed in labeled makrolon cages
- Diet: Free access to pelleted rodent diet (Kliba 3433, Provimi Kliba AG, CH-4303 Kaiseraugst)
- Water: Free access to tap water (ad libitum)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 – 25
- Humidity (%): 30 - 70
- Air changes (per hr): approx 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Treatment (epicutaneous): From: May 2, 2001 To: May 4, 2001
Treatment (intravenous): May 7, 2001
Vehicle:
other: ethanol 70%
Concentration:
0, 0.3, 3, 30 and 100%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
Not applicable (not performed).
The 4 concentrations were selected by the sponsor. The top dose is the highest level that could be achieved whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: If the results indicate a SI ≥ 3 at at least 1 test concentration and there is a dose response relationship, the test substance should be regarded as a skin sensitizer

ANIMAL ASSIGNMENT
Four groups of four animals were treated with one test substance concentration per group. One group of four animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: A weight by weight dilution was prepared using a magnetic stirrer as homogenizer. The preparation was made shortly before each dosing.
Rationale for vehicle: no data

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to OECD 429 (2002)

Observations:
Mortality/Viability: Twice daily from acclimatization to the termination of in-life phase
Body weights: At acclimatization start and prior to necropsy
Clinical signs (local/systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.
Necropsy: no necropsy was conducted
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The latest performed reliability check with Alpha-hexylcinnamicaldehyde (October 10, 2000 - October 25, 2000) indicates that the Local Lymph Node Assay was appropriately performed. SI values of 2.4, 3.7 and 7.0 were determined with the positive control at concentrations of 5, 10 and 25%, respectively.
Parameter:
SI
Value:
2.5
Test group / Remarks:
Concentration 0.3%
Remarks on result:
other: The SI values calculated for the substance concentrations 0.3, 3, 30 and 100% were 2.5, 2.2, 13.8 and 33.0 respectively.
Parameter:
SI
Value:
2.2
Test group / Remarks:
Concentration 3.0%
Remarks on result:
other: The SI values calculated for the substance concentrations 0.3, 3, 30 and 100% were 2.5, 2.2, 13.8 and 33.0 respectively
Parameter:
SI
Value:
13.8
Test group / Remarks:
Concentration 30%
Remarks on result:
other: The SI values calculated for the substance concentrations 0.3, 3, 30 and 100% were 2.5, 2.2, 13.8 and 33.0 respectively
Parameter:
SI
Value:
33
Test group / Remarks:
Concentration 100%
Remarks on result:
other: The SI values calculated for the substance concentrations 0.3, 3, 30 and 100% were 2.5, 2.2, 13.8 and 33.0 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 0.3, 3, 30 and 100% were 3033, 2584, 16501 and 39633 DPM respectively. The mean DPM/animal value for the vehicle control group was 1211 DPM.

 

Mean DPM

Mean DPM – Background (mean)

S.I. (rounded)

Background measurement 1

6

 

 

Background measurement 2

11

 

 

Vehicle

1211

1202

 

0.3%

3033

3024

2.5

3%

2584

2575

2.2

30%

16501

16492

13.8

100%

39633

39624

33.0

EC3 value

The EC3 value was not reported in the study report, but is calculated by the assessor to be 4.9% 

Other results

- No symptoms of local toxicity at the ears of the animals were observed

- No symptoms of systemic toxicity were observed

- Bodyweights were within the normal range to be expected  

- No deaths occurred during the study

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of a LLNA study, performed according to OECD 429 (2000), the substance should be considered to be a skin sensitiser, in accordance with the CLP Regulation, as the SI appeared to be ≥ 3 at 2 test concentrations and as there is a dose response relationship. The EC3 value is calculated to be 4.9%.
Executive summary:

The substance was assessed for its skin sensitization potential in female CBA/JIbm mice, using the Local Lymph Node Assay, according to OECD 429 (draft of November 2000) and in compliance with GLP. Four groups of four animals were treated with the substance at concentrations of 0.3, 3, 30 and 100% in ethanol 70%. One control group of 4 animals was treated with the vehicle alone. The latest performed reliability check indicated that the Assay was working properly. The SI values calculated for the substance concentrations 0.3, 3, 30 and 100% were 2.5, 2.2, 13.8 and 33.0 respectively. As at the 2 highest concentrations, 30 and 100%, a S.I. value of ≥ 3 was found and as there is a dose response relationship, the substance should be regarded as a skin sensitizer based on the results of this study in accordance with the CLP Regulation. No signs of irritation were observed at the ears of the animals. The EC3 value is calculated to be 4.9%.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

LLNA assay.
The substance was assessed for its skin sensitization potential in female CBA/JIbm mice, using the Local Lymph Node Assay, according to OECD 429 (draft of November 2000) and in compliance with GLP. Four groups of four animals were treated with the substance at concentrations of 0.3, 3, 30 and 100% in ethanol 70%. One control group of 4 animals was treated with the vehicle alone. The latest performed reliability check indicated that the Assay was working properly. As at the 2 highest concentrations, 30 and 100%, a S.I. value of ≥ 3 was found and as there is a dose response relationship, the substance should be regarded as a skin sensitizer based on the results of this study. The EC3 value is calculated to be 4.9%. No signs of irritation were observed at the ears of the animals.

Human repeated insult patch test
Under the conditions of a human repeated insult (occlusive) patch test procedure, a 3% solution of the substance did not show any evidence of induced allergic contact dermatitis in 52 human subjects. During induction, the 3% solution was applied to the back three times per week for three succesive weeks. Sites were covered for 24 hours with occlusive patches. Repeated applications were made to the same test sites. Reactions were scored at 48 or 72 hours after each induction application according to the Draize scale. The challenge phase was initiated approximately 2 weeks after application of the final induction patch. Challenge patches were applied for 24 hours to new sites; reactions were scored 48 and 96 hours later. It was reported that it did not induce sensitisation in any of the 52 human subjects tested.

As under the conditions of a human repeated insult (occlusive) patch test procedure, a 3% solution of the substance did not show any evidence of induced allergic contact dermatitis in 52 human subjects, a specific concentration limit (SCL) of >3% can be concluded for the sensitizing hazard of this substance.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the performed LLNA study the substance needs to be classified as a skin sensitizer (sub-category 1B) in accordance with the CLP Regulation. As under the conditions of a human repeated insult (occlusive) patch test procedure, a 3% solution of the substance did not show any evidence of induced allergic contact dermatitis in 52 human subjects, a specific concentration limit (SCL) of >3% can be concluded for the sensitizing hazard of this substance.