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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland Pfalz
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Test material is the main constituent of the registered substance.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd/SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS GmbH, C/O Postfach 553, NL-5800 AN Venray
- Females nulliparous and non-pregnant: Not specified
- Age at study initiation: 8 - 12 weeks
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 17.5g – 22.3g (pretest); 18.4g – 22.8g (main test)
- Housing: Single housed
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Basel,
Switzerland, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 d befor 1st test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 45 - 65%
- Photoperiod (hrs dark / hrs light): 12 hours light from 06:00 h to 18:00 h; 12 hours darkness from 18:00 h to 06:00 h

Study design: in vivo (LLNA)

Vehicle:
other: ethanol
Remarks:
Ethanol was used as the vehicle because good solubility of the preparation was achieved. Ethanol is the preferred vehicle for application of the test substance.
Concentration:
2.5%, 10%, 25% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429
- Irritation: A pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed.; two mice were treated with test-substance (conc. 1% and 10%; each on 3 consecutive days); Signs of
local irritation were assessed on day 1, 2 and 5
- Systemic toxicity: A pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed.; two mice were treated with test-substance (conc. 1% and 10%; each on 3 consecutive days); clinical signs were assessed after 1, 2, 5d
- Ear thickness measurements: Prior to the first application; ear thickness was determined using a micrometer after 0, 2, 5d

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Randomization
- Criteria used to consider a positive response:
incorporation of 3H-thymidine at least 3-fold or greater than that determined in control mice as indicated by the stimulation index (SI ≥ 3.0)
thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of ≥ 1.5 and ≥ 1.25, respectively

TREATMENT PREPARATION AND ADMINISTRATION:

- 4 test groups (0%, 2.5%, 10% and 25% (w/w)); 5 mice per group
- Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- On study day five (about 66 to 72 hours after the last application of test substance to the ears), 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.
- The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- Afterwards: Determination of ear weight, Removal and weight determination of the lymph nodes, Preparation of cell suspension and determination of cell count, Measurement of 3H-thymidine incorporation of the lymph node cells





Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde, techn. 85%
Statistics:
Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean
values per test group and/or single animal values by the mean of the vehicle treated group.
Results for the parameter 3H-thymidine incorporation, cell count, lymph node weight and ear weight were statistically evaluated with the WILCOXON-test.

Results and discussion

Positive control results:
A concurrent positive control (reliability check) with a known sensitizer was not included in this study

In vivo (LLNA)

Resultsopen allclose all
Parameter:
EC3
Value:
13.6
Parameter:
SI
Remarks:
3H- thymidine incorporation stimulation index
Value:
4.9
Test group / Remarks:
25%
Remarks on result:
other: significant result compared to the mean of the vehicle control; biologically relevant result
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
- The concentration of 25% has to be considered to indicate a skin sensitization potential because of lymph node responses clearly exceeding the respective cutoff values in the absence of sufficiently strong ear skin irritation

DETAILS ON STIMULATION INDEX CALCULATION
- Mean values and standard deviations of the measured parameters were calculated per test
group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell
count, lymph node weight and ear weight measurements were calculated by dividing the mean
values per test group and/or single animal values by the mean of the vehicle treated group

EC3 CALCULATION :
-If applicable, the EC leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below
and above the SI if possible or by using the two nearest points below or above the SI

CLINICAL OBSERVATIONS:
Pretest:
- No signs of systemic toxicity were observed
- The animals did not show any signs of local irritation
- Moderately increased lymph node weights (10% concentration)

Main test:
- No signs of systemic toxicity in all animals
- Biologically relevant and statistically significant response in the auricular lymph node cell counts (25% concentration)
- Statistically significant increases in lymph node weights (25% and 10% concentration)
- Moderate scaling of the ear skin was observed in all animals and slight incrustation in one animal at the 25% concentration on study day 5 (post-mortem observation)

BODY WEIGHTS:
- No influence on the mean body weight in the study


Any other information on results incl. tables

Tab. 2: Stimulation indices

Test Group

Treatment

³H-thymidine incorporation

Stimulation Index1

Cell Count Stimulation Index1

Lymph Node Weight Stimulation Index1

Ear Weight Stimulation Index1

1

vehicle ethanol

1.00

 

1.00

 

1.00

 

1.00

 

2

2.5% in ethanol

1.26

 

1.10

 

1.06

 

1.02

 

3

10% in ethanol

2.41

##

1.50

#

1.47

##

1.03

 

4

25% in ethanol

4.90

##

2.31

##

1.92

##

1.23

##

1: versus mean of test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Tab. 3: 3H-thymidine incorporation, cell count and lymph node weight: test group mean value, standard deviation and stimulation index

 

Test Group

 

Treatment

³H-thymidine incorporation [DPM/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle ethanol

151.6

20.2

1.00

2

2.5% in ethanol

191.0

42.4

1.26

3

10% in ethanol

364.8

141.0

2.41 ##

4

25% in ethanol

743.0

136.5

4.90 ##

 

Test Group

 

Treatment

Cell Counts [Counts/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle ethanol

12,715,200

737,880

1.00

2

2.5% in ethanol

13,945,600

1,375,842

1.10

3

10% in ethanol

19,123,200

5,239,266

1.50 #

4

25% in ethanol

29,336,000

2,065,697

2.31 ##

 

Test Group

 

Treatment

Lymph Node Weight [mg/Lymph Node Pair]

Mean

S.D.

Stimulation Index1

1

vehicle ethanol

5.5

0.2

1.00

2

2.5% in ethanol

5.9

0.5

1.06

3

10% in ethanol

8.2

1.5

1.47 ##

4

25% in ethanol

10.7

1.2

1.92 ##

1: versus mean of test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Tab. 4: Ear weight: test group mean value, standard deviation and stimulation index

 

Test Group

 

Treatment

Ear Weight [mg/animal]

Mean

S.D.

Stimulation Index1

1

vehicle ethanol

30.6

1.2

1.00

2

2.5% in ethanol

31.3

1.1

1.02

3

10% in ethanol

31.4

1.4

1.03

4

25% in ethanol

37.6

3.1

1.23 ##

1: versus mean of test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Tab. 5: Nature and duration of local findings of individual animals

Test Group

1

Treatment

vehicle ethanol

Animal Number

1

2

3

4

5

 

-

-

-

-

-

Test Group

2

Treatment

2.5% in ethanol

Animal Number

6

7

8

9

10

 

-

-

-

-

-

Test Group

3

Treatment

10% in ethanol

Animal Number

11

12

13

14

15

 

-

-

-

-

-

Test Group

4

Treatment

25% in ethanol

Animal Number

16

17

18

19

20

scaling / moderate1

d5

d5

d5

d5

d5

incrustation / slight1

d5

-

-

-

-

1: post mortem observation

Tab. 6: Periodic positive control data

 

Project No.

 

Date of performance

 

Name of positive control substance

 

Concentrations

   

Vehicle

Stimulation Index

3H-thymidine

incorporationa

   Stimulation Index

Cell counts

 

EC 3.0

 

EC 1.5

 

58V0288/98A008

 

Jun 2016

Alpha-Hexylcinnamaldehyde, techn. 85%

 

1%, 5%, 15%

 

MEK (methyl ethyl ketone)

 

2.60, 7.08, 14.77

 

1.50, 2.21, 3.01

 

1.4

 

1.0

 

58V0288/98A009

 

Jan 2017

Alpha-Hexylcinnamaldehyde, techn. 85%

 

1%, 5%, 15%

 

MEK (methyl ethyl ketone)

 

1.56, 3.20, 9.16

 

1.22, 2.0, 3.42

 

4.5

 

2.4

 

58V0288/98A010

 

Jul 2017

Alpha-Hexylcinnamaldehyde, techn. 85%

 

1%, 5%, 15%

 

MEK (methyl ethyl ketone)

 

2.91, 4.29, 9.61

 

1.59, 2.14, 2.92

 

1.3

 

ca. 1

 

58V0288/98A011

 

Jan 2018

Alpha-Hexylcinnamaldehyde, techn. 85%

 

1%, 5%, 15%

 

MEK (methyl ethyl ketone)

 

1.59, 3.17, 7.48

 

1.27, 1.66, 2.83

 

4.6

 

3.3

 

58V0288/98A012

 

Jun 2018

Alpha-Hexylcinnamaldehyde, techn. 85%

 

1%, 5%, 15%

 

MEK (methyl ethyl ketone)

 

2.19, 4.91, 4.71

 

1.48, 2.42, 2.51

 

2.2

 

1.1

a Ratio of test group values to control group values (Stimulation index) ≥ 3.0 indicates a positive result

Tab. 7: Positive control data of further study-related experiments

 

Project No.

 

Date of performance

 Name of positive control substance

 

Concentration

 

Vehicle

Stimulation Index

3H-thymidine

incorporationa

 Stimulation Index

Cell counts

 

 

Alpha-

 

 

 

 

58V0165/16A074

 

Hexylcinnamaldehyde,

 

MEK (methyl ethyl

 

 

58V0174/16A060

Jul 2016

techn. 85%

15%

ketone)

7.99

2.24

 

58V0010/16A057

 

Sep 2016

Alpha- Hexylcinnamaldehyde,

techn. 85%

 

15%

 

MEK (methyl ethyl ketone)

 

10.97

 

2.28

 

58V0486/16A247

 

May 2017

Alpha- Hexylcinnamaldehyde,

techn. 85%

 

15%

 

MEK (methyl ethyl ketone)

 

6.70

 

2.79

 

58V0740/13A516

 

Jul 2017

Alpha- Hexylcinnamaldehyde,

techn. 85%

 

15%

 

MEK (methyl ethyl ketone)

 

8.69

 

2.44

 

58V0154/17A1131

 

Jun 2018

Alpha- Hexylcinnamaldehyde,

techn. 85%

 

15%

 

MEK (methyl ethyl ketone)

 

4.57

 

2.80

a: Ratio of test group values to control group values (stimulation index) ≥ 3.0 indicates a positive result

1: = Stimulation Index (SI) calculated with historical control data for the vehicle MEK

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
It is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.
Executive summary:

The skin sensitizing potential of the test substance was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears.

Groups of 5 female CBA/CaOlaHsd mice each were treated with 2.5%, 10% and 25% (w/w) preparations of the test substance in ethanol or with the vehicle alone.

Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or the vehicle alone, applied to the dorsal surfaces of both ears on three consecutive days.

Three days after the last application, 20 μCi 3H-thymidine in 250 μL sterile saline were injected into the tail vein of the mice. About 5 hours after the 3H-thymidine injection, the mice were

sacrificed, and the auricular lymph nodes were removed. Lymph node response was evaluated measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell count and weight of

each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear, and for each animal, the weight of the

pooled punches was determined to obtain an indication of possible skin irritation.

No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25% preparation in ethanol, the test substance induced a biologically relevant

(increase to 3-fold or above of control value = stimulation index (SI) ≥ 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the

auricular lymph nodes. The increase of the 10% test-substance preparation was statistically significant but failed to reach the cut-off value. Concomitantly, the 25% test-substance preparation induced a biologically relevant (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) and statistically significant

response in the auricular lymph node cell counts. The SI of the 10% test-substance preparation lies at the border of biological relevance and was statistically significant.

In addition, statistically significant increases in lymph node weights were noted at the 25% and 10% concentration. The test-substance concentrations did not cause relevant increases in ear weights (SI ≥ 1.25),

demonstrating the absence of excessive ear skin irritation. However, statistically significant and considerably increased ear weight (mean SI 1.23) was noted at the 25% concentration. In

addition, moderate scaling of the ear skin was observed in all animals and slight incrustation in one animal at the 25% concentration on study day 5 (post-mortem observation).

Nevertheless, the concentration of 25% has to be considered to indicate a skin sensitization potential because of lymph node responses clearly exceeding the respective cutoff values in

the absence of sufficiently strong ear skin irritation. Thus, it is concluded that Dodecatrienal exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >10% <25% for 3H-thymidine incorporation. The EC 3 (estimated concentration that leads to the SI of 3.0) was calculated by

linear regression from the results of these concentrations to be 13.6%. The EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of all concentrations to be 10.0%.