Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

Skin sensitisation

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

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland Pfalz

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

EC Number:

701-295-5

Molecular formula:

C12 H18 O

IUPAC Name:

Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8
- Purity: Around 92 area-% as isomeric mixture determined by GC on an RTX-1701 capillary
- Physical state / color: Liquid / colorless, clear
- Expiry date: May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room tempreature
- Storage stability: Guaranteed by the sponsor

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application.
- After stirring with a magnetic stirrer, the test substance was soluble in the vehicle

FORM AS APPLIED IN THE TEST: In solution

OTHER SPECIFICS:
- Homogeneity: Homogeneous by visual inspection

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)

Results

Results 1

Parameter:

EC3

Value:

13.6

Results 2

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 Index^1		Cell Count Stimulation Index^1		Lymph Node Weight Stimulation Index^1		Ear Weight Stimulation Index^1
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 Index^1
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 Index^1
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 Index^1
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 Index^1
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 / moderate^1	d5	d5	d5	d5	d5
incrustation / slight^1	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 incorporation^a	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 incorporation^a	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
58V0154/17A113^1	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%.