1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August - 10 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22th 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

Constituent 1

Reference substance name:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

EC Number:

235-521-4

EC Name:

1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)-, reaction products with sodium sulfide (Na2(Sx)), leuco derivatives

Cas Number:

12262-32-7

Molecular formula:

not applicable

IUPAC Name:

Reaction product of 1-naphthalenesulfonic acid, 5-[(4-hydroxyphenyl)amino]-8-(phenylamino)- with sodium polysulfide, leuco derivatives

Test material form:

solid

Specific details on test material used for the study:

Date of production: 26.03.2015
Expiration date: 26.03.2020

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:

TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Hygienic level at arrival was SPF; Hygienic level during the study was good conventional.
- Age at study initiation: Young adult mice; 9-11 weeks old (at start of the main test)
- Weight at study initiation: 18.2 – 23.0 g (the weight variation in animals involved in the study did not exceed 20 % of the mean weight)
- Housing during the test: Grouped caging (4 animals/cage) in Type II cages
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum
- Water: tap water from watering bottles ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12 (from 6.00 a.m. to 6.00 p.m.)

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

1 %, 0.5 %, 0.25 % or 0.1 % (w/v)

No. of animals per dose:

4 animals/treatment group; 28 animals/main test

Details on study design:

Dose Range Finding Test
The pre-experiments on formulation evaluation and the Dose Range Finding Test (DRF) were not performed in compliance with the GLP-Regulations and are excluded from the Statement of the Study Director, but the raw data of these tests are archived under the study code of present study. The DRF was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used. All animals were observed for any clinical signs of systemic toxicity or local irritation at the application site during the preliminary test. Body weights were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both ears of each mouse were observed for erythema and scored according to criteria. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. The maximum test concentration was selected according to results of the preliminary formulation evaluation. The test item was examined in the DRF as 1 %, 0.5 % or 0.25 % (w/v) formulations prepared with the selected vehicle of DMF. All formulations were adequately homogeneous (apparently solutions, observed by the naked eye) during the application: no insoluble particles were observed although dark green colour of the formulations made the observation difficult.
Three groups of 2 CBA/Ca mice were treated with the appropriate formulations. No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed. Although body weight decreased by 5 % (1/2 animals) was observed in the 1 % (w/v) dose group it was considered neither significant nor treatment related as the mean body weight did not decrease significantly. No sign of significant irritation (indicated by an erythema score of above 3 and/or an increase of  25 % of ear thickness observed on any day of measurement) or any other local effect were observed.
Based on the preliminary test results the maximum attainable concentration (based on solubility) of 1 % (w/v) was used in the main test with the aim of testing the highest concentration possible. The test item was tested also at three additional, lower concentrations (0.5 %, 0.25 % and 0.1 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

Main Test Design
Animals in the treatment groups were treated with the negative (vehicle) controls (DMF or AOO), 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. 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 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 (Tri-Carb 3100TR, Liquid Scintillation Analyzer, 072971). 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:

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 no EC3 value (dose calculated to induce a stimulation index of 3) was calculated for the test item.

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 = 13.8) thus confirming the validity of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

2.6

Variability:

p = 0.22, r = 0.78

Test group / Remarks:

at test item concentration of 1 °%,

Results 2

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

SI values were below 3 for all tested concentrations

Results 3

Parameter:

SI

Value:

2

Variability:

p = 0.22, r = 0.78

Test group / Remarks:

at test item concentration of 0.5 °%,

Results 4

Parameter:

SI

Value:

2.3

Variability:

p = 0.22, r = 0.78

Test group / Remarks:

at test item concentration of 0.25 °%

Results 5

Parameter:

SI

Value:

1.3

Variability:

p = 0.22, r = 0.78

Test group / Remarks:

at test item concentration of 0.1 °%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No significantly increased lymphoproliferation (indicated by an SI > 3) compared to the relevant control (DMF) was noted at the applied test concentrations.

DETAILS ON STIMULATION INDEX CALCULATION
T he observed stimulation index values were 2.6, 2.0, 2.3 and 1.3 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.22, r = 0.78; evaluated by linear regression using SI values).

CLINICAL OBSERVATIONS/BODY WEIGHTS:
No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

Any other information on results incl. tables

Table 3: 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 (%)
404	Leuco Sulfur Green 2	17.5	16.6	-5
440	1 % in DMF	18.5	18.1	-2
	Mean	18.0	17.4	-4
405	Leuco Sulfur Green 2	18.4	19.2	4
441	0.5 % in DMF	17.3	16.8	-3
	Mean	17.9	18.0	1
406	Leuco Sulfur Green 2	18.5	18.9	2
442	0.25 % in DMF	17.4	18.3	5
	Mean	18.0	18.6	4

 Terminal body weights were measured on Day 6.

DMF = N,N-Dimethylformamide

Table 4: 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
C. I. Leuco Sulfur Green 2 1 % in DMF	404	N	N	N	N	N	N	N	N	N
	440	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 0.5 % in DMF	405	N	N	N	N	N	N	N	N	N
	441	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 0.25 % in DMF	406	N	N	N	N	N	N	N	N	N
	442	N	N	N	N	N	N	N	N	N

PT = Prior to the treatment

AT = After the treatment

DMF =N,N-Dimethylformamide

N = Normal (no sign of toxicity observed)

Table 5: 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
C. I. Leuco Sulfur Green 2 1 % in DMF	404	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	440	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
C. I. Leuco Sulfur Green 2 0.5 % in DMF	405	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	441	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
C. I. Leuco Sulfur Green 2 0.25 % in DMF	406	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	442	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

DMF =N,N-Dimethylformamide

Table 6: 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
	404	L	0.20	0.21	5.0	0.20	0.0
Leuco Sulfur Green 2		R	0.20	0.21	5.0	0.20	0.0
1 % in DMF	440	L	0.20	0.20	0.0	0.20	0.0
		R	0.20	0.20	0.0	0.20	0.0
	405	L	0.20	0.21	5.0	0.20	0.0
Leuco Sulfur Green 2		R	0.20	0.21	5.0	0.20	0.0
0.5 % in DMF	441	L	0.20	0.20	0.0	0.21	5.0
		R	0.21	0.20	-4.8	0.21	0.0
	406	L	0.20	0.20	0.0	0.21	5.0
Leuco Sulfur Green 2		R	0.20	0.20	0.0	0.21	5.0
0.25 % in DMF	442	L	0.20	0.21	5.0	0.21	5.0
		R	0.20	0.21	5.0	0.21	5.0

L = Left

R = Right

DMF = N,N-Dimethylformamide

 

* 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.

Table 7: Individual Body Weights of the Animals with Group Means, the Associated Error Terms and Body Weight Changes in the Main Test

Animal	Dose Group	Initial	Terminal	Body Weight
Number		Body Weight	Body Weight	Change
		(g)	(g)	(%)
461	Vehicle control for the positive control:	19.8	20.2	2
473	AOO	21.0	22.1	5
474		18.6	18.5	-1
522		22.2	22.9	3
	Mean	20.4	20.9	3
	SD	1.5	2.0
462	Positive control:	19.5	20.8	7
475	25 % HCA	20.8	21.5	3
476	in AOO	20.1	20.8	3
523		22.3	23.5	5
	Mean	20.7	21.7	5
	SD	1.2	1.3
463	Vehicle control for the test item:	19.3	20.2	5
477	DMF	21.1	21.7	3
524		23.0	22.7	-1
525		20.2	20.5	1
	Mean	20.9	21.3	2
	SD	1.6	1.2
468	C. I. Leuco Sulfur Green 2	22.8	22.5	-1
507	1 %	19.9	22.1	11
508	in DMF	19.1	19.9	4
544		20.5	22.5	10
	Mean	20.6	21.8	6
	SD	1.6	1.2
469	C. I. Leuco Sulfur Green 2	19.0	20.4	7
509	0.5 %	19.7	19.9	1
510	in DMF	22.7	22.3	-2
545		20.5	20.6	0
	Mean	20.5	20.8	2
	SD	1.6	1.0
470	C. I. Leuco Sulfur Green 2	19.9	21.6	9
511	0.25 %	20.7	19.8	-4
546	in DMF	19.2	19.8	3
547		22.4	22.8	2
	Mean	20.6	21.0	2
	SD	1.4	1.5
512	C. I. Leuco Sulfur Green 2	19.6	20.5	5
513	0.1 %	21.4	20.7	-3
548	in DMF	18.2	18.6	2
549		21.7	23.8	10
	Mean	20.2	20.9	3
	SD	1.6	2.2

 HCA =a-Hexylcinnamaldehyde

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

DMF = N,N-Dimethylformamide

Table 8: 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	461	N	N	N	N	N	N	N	N	N
	473	N	N	N	N	N	N	N	N	N
	474	N	N	N	N	N	N	N	N	N
	522	N	N	N	N	N	N	N	N	N
Positive control: 25 % HCA in AOO	462	N	N	N	N	N	N	N	N	N
	475	N	N	N	N	N	N	N	N	N
	476	N	N	N	N	N	N	N	N	N
	523	N	N	N	N	N	N	N	N	N
Vehicle control for the test item: DMF	463	N	N	N	N	N	N	N	N	N
	477	N	N	N	N	N	N	N	N	N
	524	N	N	N	N	N	N	N	N	N
	525	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 1 % in DMF	468	N	N	N	N	N	N	N	N	N
	507	N	N	N	N	N	N	N	N	N
	508	N	N	N	N	N	N	N	N	N
	544	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 0.5 % in DMF	469	N	N	N	N	N	N	N	N	N
	509	N	N	N	N	N	N	N	N	N
	510	N	N	N	N	N	N	N	N	N
	545	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 0.25 % in DMF	470	N	N	N	N	N	N	N	N	N
	511	N	N	N	N	N	N	N	N	N
	546	N	N	N	N	N	N	N	N	N
	547	N	N	N	N	N	N	N	N	N
C. I. Leuco Sulfur Green 2 0.1 % in DMF	512	N	N	N	N	N	N	N	N	N
	513	N	N	N	N	N	N	N	N	N
	548	N	N	N	N	N	N	N	N	N
	549	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)

DMF =N,N-Dimethylformamide

N = Normal (no symptoms observed)

Table 9: 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	461	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	473	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	474	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	522	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	462	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	475	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	476	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	523	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: DMF	463	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	477	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	524	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	525	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

DMF = N,N-Dimethylformamide

HCA = a-Hexylcinnamaldehyde

Table 10: Erythmia Scores in the Main Test

Dose Group	Animal Number	Ears	Days
			1		2		3		4	5	6
			PT	AT	PT	AT	PT	AT
C. I. Leuco Sulfur Green 2 1 % in DMF	468	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	507	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	508	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	544	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
C. I. Leuco Sulfur Green 2 0.5 % in DMF	469	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	509	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	510	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	545	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
C. I. Leuco Sulfur Green 2 0.25 % in DMF	470	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	511	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	546	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	547	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
C. I. Leuco Sulfur Green 2 0.1 % in DMF	512	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	513	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	548	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	549	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

DMF =N,N-Dimethylformamide

Table 11: 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	461	N
	473	N
	474	N
	522	N
Positive control: 25 % HCA in AOO	462	Larger than the relevant control (AOO)
	475	Larger than the relevant control (AOO)
	476	Larger than the relevant control (AOO)
	523	Larger than the relevant control (AOO)
Vehicle control for the test item: DMF	463	N
	477	N
	524	N
	525	N
C. I. Leuco Sulfur Green 2 1 % in DMF	468	N
	507	N
	508	N
	544	N
C. I. Leuco Sulfur Green 2 0.5 % in DMF	469	N
	509	N
	510	N
	545	N
C. I. Leuco Sulfur Green 2 0.25 % in DMF	470	N
	511	N
	546	N
	547	N
C. I. Leuco Sulfur Green 2 0.1 % in DMF	512	N
	513	N
	548	N
	549	N

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

HCA = a-Hexylcinnamaldehyde

DMF = N,N-Dimethylformamide

N = Normal

Table 12: 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:	1745	1703.5	425.9	1.0
AOO
Positive control:	23627	23585.5	5896.4	13.8
25 % HCA in AOO
Vehicle control for the test item:	1571	1529.5	382.4	1.0
DMF
C. I. Leuco Sulfur Green 2	4001	3959.5	989.9	2.6
1 % in DMF
C. I. Leuco Sulfur Green 2	3099	3057.5	764.4	2.0
0.5 % in DMF
C. I. Leuco Sulfur Green 2	3529	3487.5	871.9	2.3
0.25 % in DMF
C. I. Leuco Sulfur Green 2	2085	2043.5	510.9	1.3
0.1 % in DMF

 HCA = a-Hexylcinnamaldehyde

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

DMF =N,N-Dimethylformamide

 

*Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 41.5

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo skin sensitisation assay (LLNA) according to OECD guideline 429, the SI values for all tested concentrations were below 3. Therefore the test item is not a skin sensitizer.

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. Preliminary tests were performed to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on the results the test item was formulated in N,N-Dimethylformamide (DMF). The maximum achievable concentration (based on solubility) was 1 % (w/v). According to the results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 1 %, 0.5 %, 0.25 % or 0.1 % (w/v) formulations in DMF. All formulations were homogeneous during the application. No insoluble particles were observed although dark green colour of the formulations made the observation difficult.

Appropriate positive control (a-Hexylcinnamaldehyde, HCA), 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 above the relevant control values (SI = 13.8) thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect 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 (DMF) was noted at the applied test concentrations. The observed stimulation index values were 2.6, 2.0, 2.3 and 1.3 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.22, r = 0.78; evaluated by linear regression using SI values).

According to 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 1 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered as evidences that the test item is not a skin sensitizer.