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Description of key information

Skin Sensitisation: Sensitising (Category 1A); OECD 429; Pooles, A. (2018).

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:
24 October - 27 november 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with OECD guidance and GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No. 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands and Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood flake bedding.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: ≥ 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): RH = 30-70 %
- Air changes (per hr): ≥15/h
- Photoperiod (hrs dark / hrs light): 12:12 h
- IN-LIFE DATES: Not reported
Vehicle:
unchanged (no vehicle)
Remarks:
Acetone: olive oil (4: 1 v/v) was used to prepare 50%, 10%, 5%, 2.5% or 1% dilutions
Concentration:
Prelim = 100 (undiluted), 50, 10, 5, 2.5 and 1 % v/v (in acetone:olive oil 4:1 v/v)
Main test = 2.5, 1 and 0.5 % v/v (in acetone:olive oil v/v)
No. of animals per dose:
Prelim = 1 mouse/ concentration
Main test = 4 mice/ concentration
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Test item formulated in acetone:olive oil 4:1 v/v - homogeneity not confirmed.
- The animals treated with the undiluted test item or the test item at concentrations of 50 % or 10 % v/v in acetone/olive oil 4:1, were humanely killed, on Days 4 or 5, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project License. Hunched posture and emaciation were noted in the animals treated with the test item at concentrations of 50 % or 10 % v/v in acetone/olive oil 4:1. Body weight loss was also noted.
A greater than 25 % increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 5 % v/v in acetone/olive oil 4:1.
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted in the animals treated with the test item at concentrations of 2.5 % or 1% v/v in acetone/olive oil 4:1 .
Based on this information the dose levels selected for the main test were 2.5 %, 1 % and 0.5 % v/v in acetone/olive oil 4:1

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: LSC of pooled lymph node cells 3 days post administration of 3H-methylthymidine. The administration of 3H-methylthymidine was injected into the tail vein of the mice 5 days after the topical application of the test formulation. The stimulation index and EC3 values were determined from the results of the LSC analysis.

TREATMENT PREPARATION AND ADMINISTRATION:
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
N/A
Positive control results:
A 25 % formulation of α-Hexylcinnamaldehyde, tech., 85% illicited a stimulation index = 5.54, indicative of a positive result for sensitisation, thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
1.28
Test group / Remarks:
0.5 % v/v / Negative
Key result
Parameter:
SI
Value:
1.85
Test group / Remarks:
1 % v/v / Negative
Key result
Parameter:
SI
Value:
4.26
Test group / Remarks:
2.5 % v/v / Positive
Key result
Parameter:
EC3
Value:
1.7
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION :

See Table 4

EC3 CALCULATION :

EC3 = 1 + [[(3 - 1.85) / (4.26 - 1.85)] x (2.5 - 1)] = 1.7

CLINICAL OBSERVATIONS:

Prelim test = The animals treated with the undiluted test item or the test item at concentrations of 50% or 10% v/v in acetone/olive oil 4:1, were humanely killed, on Days 4 or 5, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project License. Hunched posture and emaciation were noted in the animals treated with the test item at concentrations of 50% or 10% v/v in acetone/olive oil 4:1.
Main test = There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS:

Prelim test = Body weight loss was noted in the 100, 50 and 10 % dose groups.
Main test = Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Table1       Prelim test – clinical observations, body weight and mortality data

 

Concentration (% v/v) in acetone/olive oil 4:1

Animal number

Body weight

(g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-dose

Post dose

Pre-dose

Post dose

Pre-dose

Post dose

100

S1

20.2

-

0

0

0

0

0

0

0

HEmK

(16.5)

-

50

S2

20.3

-

0

0

0

0

0

0

0

HEmK

(16.0)

-

10

S3

19.9

-

0

0

0

0

0

0

K

(16.3)

-

-

5

S4

19.7

19.2

0

0

0

0

0

0

0

0

0

2.5

S5

19.4

19.2

0

0

0

0

0

0

0

0

0

1

S6

21.4

21.2

0

0

0

0

0

0

0

0

0

0 = no sign of systemic toxicity

Em = emaciation

H = hunched posture

K = animal humanely killed due to occurrence of clinical signs of toxicity that approached the moderate severity level

() = bodyweight at termination

- = no data, animal dead

 

Table 2       Prelim test – local skin irritation

 

Concentration (% v/v) in acetone/olive oil 4:1

Animal number

Local skin irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

100

S1

0

0

0

0

0

0

0

0

0

0

-

-

50

S2

0

0

0

0

0

0

0

0

-

-

-

-

10

S3

0

0

0

0

0

0

0

0

-

-

-

-

5

S4

0

0

0

0

0

0

0

0

0

0

0

0

2.5

S5

0

0

0

0

0

0

0

0

0

0

0

0

1

S6

0

0

0

0

0

0

0

0

0

0

0

0

- no data, animal dead

Table 3       Prelim test – ear thickness

 

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

100 %

S1

0.22

0.22

0.23

0.22

-

-

Overall thickness (mm)

0.220

0.225

-

Overall mean ear thickness change (%)

N/A

2.273

-

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

50 %

S2

0.21

0.22

0.24

0.23

-

-

Overall thickness (mm)

0.215

0.235

-

Overall mean ear thickness change (%)

N/A

9.302

-

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

10 %

S3

0.21

0.21

0.22

0.21

-

-

Overall thickness (mm)

0.210

0.215

-

Overall mean ear thickness change (%)

N/A

2.381

-

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

5 %

S4

0.21

0.21

0.22

0.23

0.27

0.26

Overall thickness (mm)

0.210

0.225

0.265

Overall mean ear thickness change (%)

N/A

7.143

26.190

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

2.5 %

S5

0.22

0.23

0.23

0.24

0.25

0.22

Overall thickness (mm)

0.225

0.235

0.235

Overall mean ear thickness change (%)

N/A

4.444

4.444

Concentration

Animal number

Ear thickness (mm)

Day 1

Day 3

Day 6

Pre-dose

Post dose

Left

Right

Left

Right

Left

Right

1 %

S6

0.23

0.22

0.21

0.22

0.22

0.21

Overall thickness (mm)

0.225

0.215

0.215

Overall mean ear thickness change (%)

N/A

-4.444

-4.444

N/A = not applicable

 

Table 4       Main test – DPM, DPM/node and stimulation index

 

Concentration (% v/v) in acetone/olive oil 4:1

DPM

DPM/node

Stimulation index

Result

Vehicle

13624.59

1703.07

N/A

N/A

0.5

17500.67

2187.58

1.28

Negative

1

25163.78

3145.47

1.85

Negative

2.5

58049.35

7256.17

4.26

Positive

DPM = disintegrations per minute

DPM/node = Total DPM / 8 (i.e. no. of nodes = 8)

Stimulation index ≥ 3.0 = positive result

N/A = not applicable

 

Table 5       Main test – Individual clinical observations, mortality and body weight data

 

Concentration (% v/v) in acetone/olive oil 4:1

Animal number

Body weight

(g)

Day

1

2

3

4

5

6

Day 1

Day 6

Change (g)

Pre-dose

Post dose

Pre-dose

Post dose

Pre-dose

Post dose

Vehicle

1-1

19.2

19.3

0.1

0

0

0

0

0

0

0

0

0

1-2

19.3

21.2

1.9

0

0

0

0

0

0

0

0

0

1-3

18.4

20.0

1.6

0

0

0

0

0

0

0

0

0

1-4

18.5

18.6

0.1

0

0

0

0

0

0

0

0

0

0.5

2-1

20.3

20.6

0.3

0

0

0

0

0

0

0

0

0

2-2

19.0

20.3

1.3

0

0

0

0

0

0

0

0

0

2-3

18.9

21.0

2.1

0

0

0

0

0

0

0

0

0

2-4

17.6

18.4

0.8

0

0

0

0

0

0

0

0

0

1

3-1

19.7

20.4

0.7

0

0

0

0

0

0

0

0

0

3-2

21.1

20.9

-0.2

0

0

0

0

0

0

0

0

0

3-3

19.3

18.9

-0.4

0

0

0

0

0

0

0

0

0

3-4

17.6

18.6

1.0

0

0

0

0

0

0

0

0

0

2.5

4-1

18.6

19.2

0.6

0

0

0

0

0

0

0

0

0

4-2

18.3

18.6

0.3

0

0

0

0

0

0

0

0

0

4-3

19.2

21.1

2.0

0

0

0

0

0

0

0

0

0

4-4

18.8

18.6

-0.2

0

0

0

0

0

0

0

0

0

0 = no sign of systemic toxicity

 

 

 

 

 

 

 

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The test item was considered to be a sensitiser (Cat. 1A) under the conditions of the test according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008.
Executive summary:

OECD 429 (2018) - In a dermal sensitisation study, the test item was evaluated with young female adult mice (CBA/Ca) using the Local Lymph Node Assay (LLNA) in accordance with OECD 429 guidance and GLP.

 

In a preliminary screening test, conducted at test item concentrations of 100, 50, 10, 5, 2.5 and 1 % v/v, signs of clinical toxicity and body weight loss were noted at 100, 50 and 10 % v/v, resulting in the test animals being humanely killed. The mean ear thickness increased by >25 % during the study period (comparison of Day 1-6 ear thicknesses) in the 5 % v/v test group, indicating excessive irritation. The concentrations for the main test were therefore set to 0.5, 1 and 2.5 % v/v.

 

In the main test, three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in the vehicle (acetone/ olive oil 4:1 v/v). at the aforementioned test concentrations. A further group of four animals was treated with the vehicle alone. A concurrent positive control test, using a group of four animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity or local skin irritation observed. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the study. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

The test item elicited a positive response (Stimulation Index ≥ 3) when tested at a concentration 2.5 % v/v. The test item was considered to be a sensitiser under the conditions of the test with a calculated EC3 value of 1.7. Both the vehicle and positive control group animals met the validity criteria of the OECD 429 guidance document.

 

Based on the condition of this study, the test item met the criteria for classification as a sensitiser (Category 1A) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation

OECD 429 (2018) - In a dermal sensitisation study, the test item was evaluated with young female adult mice (CBA/Ca) using the Local Lymph Node Assay (LLNA) in accordance with OECD 429 guidance and GLP.

 

In a preliminary screening test, conducted at test item concentrations of 100, 50, 10, 5, 2.5 and 1 % v/v, signs of clinical toxicity and body weight loss were noted at 100, 50 and 10 % v/v, resulting in the test animals being humanely killed. The mean ear thickness increased by >25 % during the study period (comparison of Day 1-6 ear thicknesses) in the 5 % v/v test group, indicating excessive irritation. The concentrations for the main test were therefore set to 0.5, 1 and 2.5 % v/v.

 

In the main test, three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in the vehicle (acetone/ olive oil 4:1 v/v). at the aforementioned test concentrations. A further group of four animals was treated with the vehicle alone. A concurrent positive control test, using a group of four animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity or local skin irritation observed. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the study. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

The test item elicited a positive response (Stimulation Index ≥ 3) when tested at a concentration 2.5 % v/v. The test item was considered to be a sensitiser under the conditions of the test with a calculated EC3 value of 1.7. Both the vehicle and positive control group animals met the validity criteria of the OECD 429 guidance document.

 

Based on the condition of this study, the test item met the criteria for classification as a sensitiser (Category 1A) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation - The test item elicited a sensitisation index ≥ 3 at a test item concentration of 2.5 % v/v with an SI value of 4.26 observed. The resulting calculated EC3 value was 1.7. Therefore, the test item meets the criteria for classification as a sensitiser (category 1A) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.