Reaction product of 4-aminophenol with 2-ethyl-6-methylbenzenamine, sodium polysulfide and sodium metabisulfite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 December 2017 - 30 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

20 July 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine, sodium polysulfide and sodium metabisulfite

EC Number:

600-520-3

Cas Number:

1040874-53-0

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 4-​aminophenol with 2-​ethyl-​6-​methylbenzenamine, sodium polysulfide and sodium metabisulfite

Test material form:

solid: particulate/powder

Details on test material:

Test item: Blue TBR
Appearance: black bluish powder
CAS No: 1040874-53-0

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd mice

Sex:

female

Details on test animals and environmental conditions:

Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test (4 animals/treatment group and 12 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 11 weeks old (at start of the main test)
Body weight rangeat starting: 19.0 – 23.4 g; The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
Acclimatization time: 7 days

Husbandry

Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

The same conditions were used for the dose range finding and main test animals. There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.

Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

other: Pluronic®PE 9200

Concentration:

25 %, 10 %, 5 % and 2.5 %

No. of animals per dose:

28 animals/main test (4 animals/treatment group)

Details on study design:

Animals in the treatment groups were treated with the negative controls (vehicles), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.

In vivo Treatment
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
 
Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps.Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the beta-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.

Instrument used for the measurement:
Name: Tri-Carb 3100TR, Liquid Scintillation Analyzer
Serial Number: 072971

Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item.

Interpretation of the Results
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The randomization was checked by computer software [SPSS/PC+ (4.0.1)]

Results and discussion

Positive control results:

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 9.5) thus confirming the validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

p = 0.36, r = 0.64

Test group / Remarks:

test item concentration of 25 %

Results 2

Key result

Parameter:

SI

Value:

0.5

Variability:

p = 0.36, r = 0.64

Test group / Remarks:

test item concentration of 10 %,

Results 3

Key result

Parameter:

SI

Value:

0.5

Variability:

p = 0.36, r = 0.64

Test group / Remarks:

at test item concentration of 5 %

Results 4

Key result

Parameter:

SI

Value:

0.8

Variability:

p = 0.36, r = 0.64

Test group / Remarks:

at test item concentration of 2.5 %

Cellular proliferation data / Observations:

No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effects on body weights were observed in any of the dose groups. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for Blue TBR at the applied test concentrations. The observed stimulation index values were 1.0, 0.5, 0.5 and 0.8 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.36, r = 0.64; evaluated by linear regression using SI values). According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 25 % (w/w) as well as the lack of a significant dose-related response are considered as evidence that Blue TBR is not a skin sensitizer.

Any other information on results incl. tables

Individual Body Weights of the Animals with Group Means and the Body Weight Changes in the Dose Range Finding Test

Animal	Dose Group	Initial Body	Terminal Body	Body Weight
Number		Weight (g)	Weight (g)*	Change (%)
27	Blue TBR	18.3	20.9	14
28	25 % in Plu	19.3	22.0	14
	Mean	18.8	21.5	14
29	Blue TBR	19.2	22.2	16
30	10 % in Plu	18.0	19.3	7
	Mean	18.6	20.8	12

 

*Terminal body weights were measured on Day 6.

 

Clinical Observations in the Dose Range Finding Test

Dose Group	Animal Number	Days
		1		2		3		4	5	6
		PT	AT	PT	AT	PT	AT
Blue TBR 25 % in Plu	27	N	N	N	N	N	N	N	N	N
	28	N	N	N	N	N	N	N	N	N
Blue TBR 10 % in Plu	29	N	N	N	N	N	N	N	N	N
	30	N	N	N	N	N	N	N	N	N

PT = Prior to the treatment

AT = After the treatment

Plu= aqueous 1 % (w/v) Pluronic®PE 9200

N = Normal (no sign of toxicity observed)

 

 

Erythema Scores in the Dose Range Finding Test

Dose Group	Animal Number	Ears	Days
			1		2		3		4	5	6
			PT	AT	PT	AT	PT	AT
Blue TBR 25 % in Plu	27	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	28	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Blue TBR 10 % in Plu	29	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	30	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0

L = Left                                                           R = Right

PT = Prior to the treatment                                AT = After the treatment

Plu= aqueous 1 % (w/v) Pluronic®PE 9200

Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test

Dose Group	Animal	Ears	Day 1^*	Day 3^$	Day 3	Day 6^#	Day 6
	Number		value (mm)	value (mm)	% deviation	value (mm)	% deviation
	27	L	0.20	0.20	0.0	0.19	-5.0
Blue TBR		R	0.20	0.20	0.0	0.19	-5.0
25 % in Plu	28	L	0.19	0.19	0.0	0.19	0.0
		R	0.19	0.19	0.0	0.19	0.0
	29	L	0.20	0.20	0.0	0.20	0.0
Blue TBR		R	0.20	0.20	0.0	0.20	0.0
10 % in Plu	30	L	0.19	0.20	5.3	0.20	5.3
		R	0.19	0.20	5.3	0.20	5.3

 

L = Left

R = Right

Plu= aqueous 1 % (w/v) Pluronic®PE 9200

 

* Ear thickness was measured prior to the first treatment.

$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).

# Ear thickness was measured at the end of the test.

Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test

Animal	Dose Group	Initial	Terminal	Body Weight
Number		Body Weight	Body Weight	Change
		(g)	(g)	(%)
38	Vehicle control	20.1	19.2	-4
39	for the positive control:	20.5	20.4	0
40	AOO	23.4	22.4	-4
41		22.0	21.6	-2
	Mean	21.5	20.9	-3
	SD	1.5	1.4
42	Positive control:	20.1	20.2	0
43	25 % HCA	20.8	20.4	-2
44	in AOO	21.3	20.8	-2
45		23.1	23.1	0
	Mean	21.3	21.1	-1
	SD	1.3	1.3
86	Vehicle control	21.2	21.4	1
87	for the test item:	19.9	18.9	-5
88	Plu	21.9	21.8	0
89		22.4	21.8	-3
	Mean	21.4	21.0	-2
	SD	1.1	1.4
106	Blue TBR	19.5	19.5	0
107	25 %	22.0	20.9	-5
108	in Plu	21.9	20.8	-5
109		21.1	20.6	-2
	Mean	21.1	20.5	-3
	SD	1.2	0.6
110	Blue TBR	22.1	20.8	-6
111	10 %	21.1	20.4	-3
112	in Plu	19.4	20.0	3
113		21.9	20.5	-6
	Mean	21.1	20.4	-3
	SD	1.2	0.3
114	Blue TBR	19.2	19.6	2
115	5 %	22.0	21.4	-3
116	in Plu	21.2	20.5	-3
117		21.8	20.9	-4
	Mean	21.1	20.6	-2
	SD	1.3	0.8
118	Blue TBR	21.3	20.6	-3
119	2.5 %	19.0	19.0	0
120	in Plu	22.0	21.4	-3
121		21.8	20.9	-4
	Mean	21.0	20.5	-3
	SD	1.4	1.0

HCA =a-Hexylcinnamaldehyde                           AOO = Acetone: Olive oil 4:1 (v/v) mixture

Plu= aqueous 1 % (w/v) Pluronic®PE 9200            SD = Standard Deviation

Clinical Observations in the Main Test

Dose Group	Animal Number	Days
		1		2		3		4	5	6
		PT	AT	PT	AT	PT	AT
Vehicle control for the positive control: AOO	38	N	N	N	N	N	N	N	N	N
	39	N	N	N	N	N	N	N	N	N
	40	N	N	N	N	N	N	N	N	N
	41	N	N	N	N	N	N	N	N	N
Positive control: 25 % HCA in AOO	42	N	N	N	N	N	N	N	N	N
	43	N	N	N	N	N	N	N	N	N
	44	N	N	N	N	N	N	N	N	N
	45	N	N	N	N	N	N	N	N	N
Vehicle control for the test item: Plu	86	N	N	N	N	N	N	N	N	N
	87	N	N	N	N	N	N	N	N	N
	88	N	N	N	N	N	N	N	N	N
	89	N	N	N	N	N	N	N	N	N
Blue TBR 25 % in Plu	106	N	N	N	N	N	N	N	N	N
	107	N	N	N	N	N	N	N	N	N
	108	N	N	N	N	N	N	N	N	N
	109	N	N	N	N	N	N	N	N	N
Blue TBR 10 % in Plu	110	N	N	N	N	N	N	N	N	N
	111	N	N	N	N	N	N	N	N	N
	112	N	N	N	N	N	N	N	N	N
	113	N	N	N	N	N	N	N	N	N
Blue TBR 5 % in Plu	114	N	N	N	N	N	N	N	N	N
	115	N	N	N	N	N	N	N	N	N
	116	N	N	N	N	N	N	N	N	N
	117	N	N	N	N	N	N	N	N	N
Blue TBR 2.5 % in Plu	118	N	N	N	N	N	N	N	N	N
	119	N	N	N	N	N	N	N	N	N
	120	N	N	N	N	N	N	N	N	N
	121	N	N	N	N	N	N	N	N	N

 

PT = Prior to the treatment

AT = After the treatment

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 mixture (v/v)

Plu= aqueous 1 % (w/v) Pluronic®PE 9200

 

N = Normal (no symptoms observed)

Erythema Scores in the Main Test

Dose Group	Animal Number	Ears	Days
			1		2		3		4	5	6
			PT	AT	PT	AT	PT	AT
Vehicle control for the positive control: AOO	38	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	39	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	40	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	41	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Positive control: 25 % HCA in AOO	42	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	43	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	44	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	45	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Vehicle control for the test item: Plu	86	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	87	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	88	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	89	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Blue TBR 25 % in Plu	106	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	107	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	108	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	109	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Blue TBR 10 % in Plu	110	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	111	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	112	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	113	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Blue TBR 5 % in Plu	114	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	115	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	116	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	117	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Blue TBR 2.5 % in Plu	118	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	119	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	120	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	121	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0

L = Left                                     R = Right                    PT = Prior to treatment                               AT = After the treatment

AOO = Acetone: Olive oil 4:1 (v/v) mixture                    HCA =a-Hexylcinnamaldehyde                  Plu= aqueous 1 % (w/v) Pluronic®PE 9200

Visual Observations of the Lymph Nodes in the Main Test

Dose Group	Animal Number	Appearance of Lymph Nodes
Vehicle control for the positive control: AOO	38	N
	39	N
	40	N
	41	N
Positive control: 25 % HCA in AOO	42	Larger than the relevant control (AOO)
	43	Larger than the relevant control (AOO)
	44	Larger than the relevant control (AOO)
	45	Larger than the relevant control (AOO)
Vehicle control for the test item: Plu	86	N
	87	N
	88	N
	89	N
Blue TBR 25 % in Plu	106	N
	107	N
	108	N
	109	N
Blue TBR 10 % in Plu	110	N
	111	N
	112	N
	113	N
Blue TBR 5 % in Plu	114	N
	115	N
	116	N
	117	N
Blue TBR 2.5 % in Plu	118	N
	119	N
	120	N
	121	N

 

AOO = Acetone: Olive oil 4:1 (v/v) mixture

HCA =a-Hexylcinnamaldehyde

Plu= aqueous 1 % (w/v)Pluronic®PE 9200

N = Normal

DPM and Stimulation Index Values for all Groups in the Main Test

Dose Group	Measured	Group^*	DPM/Mouse^#	Stimulation
	DPM/group	DPM		Index Values
Vehicle control for the positive control:	8360	8335.5	2083.9	1.0
AOO
Positive control:	78929	78904.5	19726.1	9.5
25 % HCA in AOO
Vehicle control for the test item:	3549	3524.5	881.1	1.0
Plu
Blue TBR	3376	3351.5	837.9	1.0
25 % in Plu
Blue TBR	1909	1884.5	471.1	0.5
10 % in Plu
Blue TBR	1853	1828.5	457.1	0.5
5 % in Plu
Blue TBR	2722	2697.5	674.4	0.8
2.5 % in Plu

 

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

Plu= aqueous 1 % (w/v) Pluronic®PE 9200

 

*Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 24.5

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present assay, the test item was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

The aim of this study according to OECD guideline 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Preliminary tests were performed to find an appropriate vehicle and the maximum applicable concentration. Test item formulations prepared in N,N-Dimethylformamide (DMF), in Dimethyl sulfoxide (DMSO) or inaqueous 1 % (w/v) Pluronic®PE 9200 (Plu) at 50 %, 25 % and 12.5 % (w/w) concentrations were evaluated. The most adequate and homogeneous formulation (apparently suspension) was achieved in Pluat at a maximum concentration of 25 % (w/w). No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test at the tested concentrations [25 % and 10 % (w/w)]. According to this the test item was examined in the main test at concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w) as suspension formulations in Plu. An appropriate positive control (a-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 9.5) thus confirming the validity of the assay. No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effects on body weights were observed in any of the dose groups. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (Plu) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.0, 0.5, 0.5 and 0.8 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.36, r = 0.64; evaluated by linear regression using SI values). According to the evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 25 % (w/w) as well as the lack of a significant dose-related response is considered as evidence that the test item is not a skin sensitizer. In conclusion, under the conditions of the present assay, the test item tested at the maximum feasible concentration of 25 % (w/w) and also at concentrations of 10 %, 5 % or 2.5 % (w/w) as adequate homogeneous formulations (suspensions, prepared with aqueous Pluronic®PE 9200 as vehicle) was shown to have no skin sensitization potential in the Local Lymph Node Assay.