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Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From August 06 to September 09, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on March 12 to 14, 2014/ signed on May 12, 2014)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were group housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014 Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From August 06 to September 09, 2014
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 10, 25 and 50 % v/v in acetone/olive oil 4:1
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- The mice were treated by daily application of 25 μL of the undiluted test item or the test item at a concentration of 50 % v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and Days 4 to 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and every Day after. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Irritation: No signs of systemic toxicity were noted. Very slight erythema (score 1) was noted on Day 3 and irritation indicated by greater than 25 % increase in ear thickness was noted on Day 5 and 6 with the mouse treated with the undiluted test item. No signs of visual irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted with the mouse treated at a concentration of 50 % v/v in acetone/olive oil 4:1. Based on this information the test item at concentrations of 10, 25 and 50 % v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. 5 h later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non-homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) were presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)
Positive control results:
A group of five animals was treated with 50 µl (25 µl per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 25 % v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone. With a SI = 12.76, α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.
Parameter:
SI
Remarks on result:
other: Stimulation index for 10, 25 and 50 % were 1.83, 3.62 and 6.02, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM / animal for vehicle, 10, 25 and 50 % were 1037.69 (± 296.73), 1902.25 (±769.92), 3753.72 (±718.01) and 6247.68 (±1237.53), respectively.

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity or excessive local skin irritation were noted in the test or control animals during the test.

 

Bodyweight

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Table 7.4.1/1:Individual Disintegrations per Minute and Stimulation Indices

Concentration

(% v/v) in

acetone/olive oil

4:1

Animal

Number

dpm/

Animal a

Mean dpm/Animal

(Standard Deviation)

Stimulation

Index b

Result

Vehicle

1-1

1260.90

1037.69

(±296.73)

NA

NA

1-2

573.57

1-3

909.11

1-4

1255.34

1-5

1189.55

10

2-1

700.44

1902.25

(±769.92)

1.83

Negative

2-2

1967.42

2-3

2085.21

2-4

1913.07

2-5

2845.09

25

3-1

4491.35

3753.72***

(±718.01)

3.62

Positive

3-2

3830.81

3-3

4409.10

3-4

2986.90

3-5

3050.44

50

4-1

7495.92

6247.68***

(±1237.53)

6.02

Positive

4-2

5852.64

4-3

7201.43

4-4

4377.36

4-5

6311.03

 

dpm = Disintegrations per minute; a = Total number of lymph nodes per animal is 2; b = Stimulation Index of 3.0 or greater indicates a positive result; na = Not applicable; *** = Significantly different from control group p<0.001

EC3 value: The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 19.80 %.
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test material is classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP).
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca (CBA/CaOlaHsd) strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

In preliminary screening test, no signs of systemic toxicity were noted. Very slight erythema (score 1) was noted on Day 3 and irritation indicated by greater than 25% increase in ear thickness was noted on Day 5 and 6 with the mouse treated with the undiluted test item. No signs of visual irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted with the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1. Based on this information the test item at concentrations of 10, 25 and 50 % v/v in acetone/olive oil 4:1 were selected for the main test.

 

Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of test material as asolution in acetone/olive oil 4:1 at concentrations of 100 %, 50 % or 25 % v/v for 3 consecutive days. A further group of five animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-Methyl Thymidine.

 

The Stimulation Index values expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration

Mean dpm/animal

Stimulation Index

Result

Vehicle

1037.69 (±296.73)

NA

N/A

10 %

1902.25 (±769.92)

1.83

Negative

25 %

3753.72 (±718.01)

3.62

Positive

50 %

6247.68 (±1237.53)

6.02

Positive

 

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 19.80 %.

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 12.76, when tested at 25 % v/v. The test system was therefore considered to be valid.

 

There were no deaths. No signs of systemic toxicity or excessive local skin irritation were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Under the test conditions, test material is classified as a skin sensitizer in the Local Lymph Node Assay according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A key study was identified (Harlan, 2014). This Local Lymph Node Assay (LLNA) was performed according to the OECD test guideline No 429 and in compliance with GLP. In the preliminary screening test, very slight erythema (score 1) was noted on Day 3 and irritation indicated by greater than 25% increase in ear thickness was noted on Day 5 and 6 with the mouse treated with the undiluted test item. No signs of visual irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted with the mouse treated at a concentration of 50% v/v in acetone/olive oil 4:1. Based on this information the test item at concentrations of 10, 25 and 50 % v/v in acetone/olive oil 4:1 were selected for the main test.

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1 to 6.

The Stimulation Index values were 1.83, 3.62 and 6.02 for the concentrations of 10%, 25% and 50%, respectively.

The concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 19.80 %.

The historical positive control,α-Hexylcinnamaldehyde, gave a SI of 12.72, when tested at 25 % v/v. The test system was therefore considered to be valid.

There were no deaths. No signs of systemic toxicity or excessive local skin irritation were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Under the test conditions, the test item is classified as a skin sensitizer.


Migrated from Short description of key information:
LLNA, sensitising (OECD 429, GLP, K, Rel. 1)

Justification for selection of skin sensitisation endpoint:
Only one study available, GLP-compliant and of high quality (Klimisch score = 1).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 including ATP6.

Self-classification:

Based on the available data, the substance is classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP), since EC3 is > 2%.

No data was available regarding respiratory sensitisation.