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Description of key information

Skin Sensitisation: Skin sensitiser (category 1B); OECD 429, Anon, 2009

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:
15 Jun - 14 Jul 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in accordance with international guidelines and in accordance with GLP. All guideline criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
Exceptions to GLP noted, but satsifactory counterpoints are provided to ensure that the study can still be considered GLP compliant
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME, USA, 04069
- Females (if applicable) nulliparous and non-pregnant: not specified (but were purpose bred and experimentally naive)
- Microbiological status of animals, when known: N/A
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 18-25g
- Housing: Group housed 5 per cage
- Diet (e.g. ad libitum): Ad libitum: Harlan Teklad Certified Rodent Chow 7012c
- Water (e.g. ad libitum): Ad libitum: tap water
- Acclimation period: Acclimated to housing 5 days prior to first day of dosing
- Indication of any skin lesions: All animals assessed for general health and only those deemed suitable were used in the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): Relative humidity: 44-62%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): 12 light/12 dark hrs
- IN-LIFE DATES: From: To: 8 July 2009 - 14 July 2009
Vehicle:
other: EtOH/DEP
Concentration:
The test article was prepared at 2.5%, 5%, 10%, 25% or 50% v/v.
The positive control was prepared daily as 5%, 15% and 35% solutions in the vehicle.
Justification for dose levels: generally, doses were selected so that the highest concentration maximised exposure whilst avoiding systemic toxicity and excessive local irritation. Doses were selected based on known reported uses of the material.
No. of animals per dose:
n=45
No. of test groups=9
No. of animals per dose (including control): 5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
- Irritation: animals were examined at least once daily for abnormalities and general health
- Systemic toxicity: N/A
- Ear thickness measurements: N/A
- Erythema scores: N/A

MAIN STUDY
-Route: Topically on the dorsal surface of both ears
Frequency: Once daily for 3 consecutive days (Days 1-3). The timing of dose administration remained consistent (±2 hours) during the dosing phase.
Procedure: A volume of 25ul/ear was applied using a micro pipette.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: N/A

TREATMENT PREPARATION AND ADMINISTRATION: The test substance was applied topically
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
SYSTAT v 9.01 developed by SPSS, Inc.
Individual DPM values were analyzed and the mean DPM plus the standard error were calculated for each group. Body weights on days 1 and 6 and body weight changes were evaluated. The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical distribution. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.
Inidivdiual DPM values were analysed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM and body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance.
Positive control results:
Clinical observations:
The positive control α- Hexylcinnamaldehyde (HCA) caused no mortality. Ears appeared wet in all treated animals Days 2-4. Ears appeared wet at 35% dose on Day 5.
LLNA results:
At termination, animals treated with 35% HCA exhibited enlarged lymph nodes. Exposure to HCA at 5%, 15% and 35% v/v resulted in stimulation indices of 1.2, 1.9 and 9.4 respectively. A 3-fold or greater increase in proliferative activity to the concurrent vehicle control is considered a positive response.
Key result
Parameter:
EC3
Value:
23.6
Parameter:
SI
Value:
0.9
Test group / Remarks:
2.5 %
Parameter:
SI
Value:
1.1
Test group / Remarks:
5 %
Parameter:
SI
Value:
1.1
Test group / Remarks:
10 %
Parameter:
SI
Value:
3.2
Test group / Remarks:
25 %
Parameter:
SI
Value:
12.4
Test group / Remarks:
50 %

Table 1 demonstrates the dermal irritation scores attributed to each test animal. The Draize (1959) definition for scoring dermal irritation was used. Each dosed test animal yielded a dermal irritation score of 0 (i.e no erythema and no edema) an all tested days. Table 2 shows the Local Lymph Node Assay results. Table 3 is a breakdown of individual animal data.

There was no mortality in all test animals.

Clinical observations:

No erythema or edema was noted in any of the mice at 2.5%, 5%, 10%, 25% or 50% dose levels. Ears appeared wet in the mice treated at doses 25% and 50% on Days 2 and 4. Ears appeared wet in mice treated with the 50% dose level on Day 5. The mean body weights and body weight changes of mice treated with LLNA-641 on Days 1 and 6 showed no statistically significant differences. Therefore, administration of the test item appeared not to exert overt toxicity.

LLNA results:

At termination, lymph nodes from the mice treated with 2.5%, 5%, 10% or 25% were normal in size and appearance. Exposure to LLNA-641 resulted in stimulation indices 0.9, 1.1, 1.1, 3.2 and 12.4 respectively. In addition, the responses with 25% and 50% of the test article was statistically significant (p<0.001) when the log DPM was compared to the vehicle group. Since the data indicated a positive response the EC3 was calculated (see below equation) and determined to be 23.6%.

EC3 = c+[(3-d)/(b-d)](a-c)

where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Exposure to HCA at 5%, 15% and 35% (v/v) resulted in stimulation indices of 1.2, 1.9 and 9.4, respectively. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response.

2.5%, 5%, 10% or 25%

Table 1 Dermal irritation scores

MOUSE NO.

GROUP

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

Erythema

Edema

1

1

0

0

0

0

0

0

0

0

0

0

0

0

2

1

0

0

0

0

0

0

0

0

0

0

0

0

3

1

0

0

0

0

0

0

0

0

0

0

0

0

4

1

0

0

0

0

0

0

0

0

0

0

0

0

5

1

0

0

0

0

0

0

0

0

0

0

0

0

6

2

0

0

0

0

0

0

0

0

0

0

0

0

7

2

0

0

0

0

0

0

0

0

0

0

0

0

8

2

0

0

0

0

0

0

0

0

0

0

0

0

9

2

0

0

0

0

0

0

0

0

0

0

0

0

10

2

0

0

0

0

0

0

0

0

0

0

0

0

11

3

0

0

0

0

0

0

0

0

0

0

0

0

12

3

0

0

0

0

0

0

0

0

0

0

0

0

13

3

0

0

0

0

0

0

0

0

0

0

0

0

14

3

0

0

0

0

0

0

0

0

0

0

0

0

15

3

0

0

0

0

0

0

0

0

0

0

0

0

16

4

0

0

0

0

0

0

0

0

0

0

0

0

17

4

0

0

0

0

0

0

0

0

0

0

0

0

18

4

0

0

0

0

0

0

0

0

0

0

0

0

19

4

0

0

0

0

0

0

0

0

0

0

0

0

20

4

0

0

0

0

0

0

0

0

0

0

0

0

21

5

0

0

0

0

0

0

0

0

0

0

0

0

22

5

0

0

0

0

0

0

0

0

0

0

0

0

23

5

0

0

0

0

0

0

0

0

0

0

0

0

24

5

0

0

0

0

0

0

0

0

0

0

0

0

25

5

0

0

0

0

0

0

0

0

0

0

0

0

26

6

0

0

0

0

0

0

0

0

0

0

0

0

27

6

0

0

0

0

0

0

0

0

0

0

0

0

28

6

0

0

0

0

0

0

0

0

0

0

0

0

29

6

0

0

0

0

0

0

0

0

0

0

0

0

30

6

0

0

0

0

0

0

0

0

0

0

0

0

31

7

0

0

0

0

0

0

0

0

0

0

0

0

32

7

0

0

0

0

0

0

0

0

0

0

0

0

33

7

0

0

0

0

0

0

0

0

0

0

0

0

34

7

0

0

0

0

0

0

0

0

0

0

0

0

35

7

0

0

0

0

0

0

0

0

0

0

0

0

0 = no (erythema/edema)

Table 2. Local Lymph Node Assay

GROUP

TREATMENT

DOSE

DPM

SI(Test/control Ratio)

RESULTS*

1

DEP/EtOH

-

320.7±49.7

-

-

2

LLNA-641

2.5%

295.2±41.3

0.9

-

3

LLNA-641

5%

358.4±64.8

1.1

-

4

LLNA-641

10%

351.2±57.4

1.1

-

5

LLNA-641

25%

1035.5±138.7**

3.2

+

6

LLNA-641

50%

3968.6±855.3**

12.4

+

7

HCA

5%

38.4±114.5

1.2

-

8

HCA

15%

602.4±117.2

1.9

-

9

HCA

35%

3027.4±208.9**

9.4

+

*test/control ratio of 3.0 or greater represents a positive result

**Statistically significant difference when log DPM compared to the vehicle control group (Group 1) (p<0.001)

Table 3. individual animal data

GROUP

ANIMAL #

DPM

LogDPM

BODY WEIGHT ON DAY 1

BODY WEIGHT ON DAY 6

CHANGE IN BODY WEIGHT

DEP/EtOH

1

258.81

2.41

25

26

1

2

322.08

2.51

25

26

1

3

169.15

2.23

21

21

0

4

428.58

2.63

20

20

0

5

425.04

2.63

22

23

1

LLNA-641 2.5%

6

182.98

2.26

22

22

0

7

397.12

2.60

24

24

0

8

214.63

2.33

24

24

0

9

356.49

2.55

23

25

2

10

325.00

2.51

25

28

3

LLNA-641 5%

11

317.04

2.50

22

22

0

12

529.33

2.72

21

21

0

13

323.94

2.51

21

20

-1

14

465.03

2.67

24

24

0

15

156.88

2.20

20

20

0

LLNA-641 10%

16

278.46

2.44

20

25

5

17

570.42

2.76

19

20

1

18

289.84

2.46

25

19

-6

19

257.53

2.41

21

22

1

20

359.55

2.56

19

19

0

LLNA-641 25%

21

1305.62

3.12

23

22

-1

22

552.08

2.74

22

21

-1

23

901.68

2.96

24

23

-1

24

1200.52

3.08

22

21

-1

25

1217.46

3.09

24

22

-2

LLNA-641 50%

26

2471.75

3.39

25

25

0

27

2404.76

3.38

23

23

0

28

4991.02

3.70

21

21

0

29

6834.54

3.83

22

23

1

30

3141.11

3.50

23

23

0

HCA 5%

31

149.61

2.17

23

24

1

32

150.88

2.18

20

21

1

33

744.57

2.87

23

22

-1

34

526.26

2.72

24

24

0

 

35

330.77

2.52

24

25

1

HCA 15%

36

910.43

2.96

21

21

0

37

688.52

2.84

21

22

1

38

219.47

2.34

18

18

0

39

483.83

2.68

22

23

1

40

709.75

2.85

20

21

1

HCA 35%

41

3046.45

3.48

19

19

0

42

3038.42

3.48

22

21

-1

43

3650.50

3.56

20

20

0

44

3068.61

3.49

22

22

0

45

2332.87

3.37

23

22

-1

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance is considered to have skin sensitising activity if, at one or more concentrations it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index of =>3.0 is regarded as a positive response. Treatment with LLNA-641 at concentrations of 25% and 50% v/v resulted in stimulation indices stimulation indices of 3 or greater and hence is considered to have skin sensitising activity. The EC3 was calculated to be 23.6% (Category 1B - moderate potency).
Executive summary:

The test substance was assessed for skin sensitisation according to OECD Testing Guidelines 429 and OPPTS Guidelines 870.2600.

The test article was prepared at 2.5%, 5%, 10%, 25% or 50% v/v. The positive control was prepared daily as 5%, 15% and 35% solutions in the test vehicle. 7 groups of 5 female CBA/J mice were treated on the dorsal surface of both ears once per day for 3 days. On Day 6, the mice were euthanised and the lymph nodes removed. There was no mortality during the experiment, and there was no erythema or edema. Ears appeared wet in the mice treated at doses 25% and 50% on Days 2 and 4. Ears appeared wet in mice treated with the 50% dose level on Day 5. The mean body weights and body weight changes of mice treated with LLNA-641 on days 1 and 6 showed no statistically significant differences. Therefore, administration of the test item appeared not to exert overt toxicity.

As per the CLP criteria, the calculated EC3 (23.6%) is greater than 2%, which designates the substance as skin sensiser category 1B.

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

The test substance was assessed for skin sensitisation according to OECD Testing Guidelines 429 and OPPTS Guidelines 870.2600.

The test article was prepared at 2.5%, 5%, 10%, 25% or 50% v/v. The positive control was prepared daily as 5%, 15% and 35% solutions in the test vehicle. 7 groups of 5 female CBA/J mice were treated on the dorsal surface of both ears once per day for 3 days. On Day 6, the mice were euthanised and the lymph nodes removed. There was no mortality during the experiment, and there was no erythema or edema. Ears appeared wet in the mice treated at doses 25% and 50% on Days 2 and 4. Ears appeared wet in mice treated with the 50% dose level on Day 5. The mean body weights and body weight changes of mice treated with LLNA-641 on days 1 and 6 showed no statistically significant differences. Therefore, administration of the test item appeared not to exert overt toxicity.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The criteria are met for classification of the substance as a skin sensitiser (skin sens. 1B) in accordance with Regulation (EC) No 1272/2008 (CLP).

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