Phenol, 2,4-dinitro-, sulfurized, thiosulfonated

Endpoint summary

Description of key information

In an in vivo local lymphnode assay (LLNA) according to OECD 429, the test item did not show skin sensitising properties.

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:

04 December, 2017 - 08 January, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

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)

Specific details on test material used for the study:

Expiry date: November 30, 2021

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd mice (SPF)

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; 11 weeks old (at start of the main test)
- Weight at study initiation: 19.1 – 23.4 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: Only healthy animals (and not showing any sign of skin lesion) were used

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.)

Vehicle:

other: Pluronic®PE 9200 (Plu)

Concentration:

25 %, 10 %, 5 % and 2.5 % (w/w) as suspension in Plu

No. of animals per dose:

4 animals/dose group

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
LLNA
- Criteria used to consider a positive response:
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.

TREATMENT PREPARATION AND ADMINISTRATION:
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. Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6. 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.

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 (ca. 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: Tri-Carb 3100TR Liquid Scintillation Analyzer, Serial Number: 072971
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)] according to the actual body weights verifying the homogeneity and deviations between the groups.

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 9.5). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

Key result

Parameter:

SI

Value:

0.5

Variability:

p = 0.63, r = 0.37

Test group / Remarks:

test item concentration of 25 %

Remarks on result:

other: No significant dose-response relationship was observed.

Key result

Parameter:

SI

Value:

0.5

Variability:

p = 0.63, r = 0.37

Test group / Remarks:

test item concentration of 10 %

Remarks on result:

other: No significant dose-response relationship was observed

Key result

Parameter:

SI

Value:

0.6

Variability:

p = 0.63, r = 0.37

Test group / Remarks:

test item concentration of 5 %.

Remarks on result:

other: No significant dose-response relationship was observed

Key result

Parameter:

SI

Value:

0.5

Variability:

p = 0.63, r = 0.37

Test group / Remarks:

at test item concentration of 2.5 % (w/w)

Remarks on result:

other: No significant dose-response relationship was observed

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No significant lymphoproliferative response (SI ≥ 3) compared to the concurrent control (Plu) was noted for Solubilised Sulphur Black 1 at the applied test concentrations. The observed stimulation index values were 0.5, 0.5, 0.6 and 0.5 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No significant dose-related response was observed (p = 0.63, r = 0.37). Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. The appearance of the lymph nodes were normal in both negative (vehicle) control groups and in the test item treated groups.

DETAILS ON STIMULATION INDEX CALCULATION
No significant lymphoproliferative response (SI > 3) compared to the relevant vehicle control (Plu) was noted for the test item at the tested concentrations. The observed stimulation index values were 0.5, 0.5, 0.6 and 0.5 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. No dose-related response was observed.

EC3 CALCULATION
not determinable

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score > 3) or any other local effect were observed in any treatment group. Dark colour of the test item formulations (black) made observation of erythema difficult on the first treatment day in the 25 % and in the 10 % (w/w) dose groups. Test item residual was also observed in the 25 % (w/w) dose group on Days 2 and 3 and in the 10 % (w/w) dose group on Day 2 (after the treatments), but it was not significant and did not interfere the observation of signs of irritation on these days. It is unlikely that an erythema of No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score > 3) occurred during the test.

BODY WEIGHTS
Body weight decrease by > 5 % was observed in the 25 % and in the 5 % (w/w) dose groups (1 of the 4 animals with 6 % decrease in both groups). The effect was not dose dependent and was not considered relevant as the mean values did not decrease significantly. No body weight decrease by > 5 % was observed in the other groups.

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 (%)
23	Solubilised Sulphur Black 1	18.5	21.3	15
24	25 % in Plu	19.5	21.4	10
	Mean	19.0	21.4	12
25	Solubilised Sulphur Black 1	18.4	21.0	14
26	10 % in Plu	19.8	21.7	10
	Mean	19.1	21.4	12

 *Terminal body weights were measured on Day 6.

 

 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
Solubilised Sulphur Black 1 25 % in Plu	23	N	N	N	N	N	N	N	N	N
	24	N	N	N	N	N	N	N	N	N
Solubilised Sulphur Black 1 10 % in Plu	25	N	N	N	N	N	N	N	N	N
	26	N	N	N	N	N	N	N	N	N

T = Treatment

PT = Prior to the treatment

AT = After the treatment

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

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
Solubilised Sulphur Black 1 25 % in Plu	23	L	0	NO	0	NO	0	NO	0	0	0
		R	0	NO	0	NO	0	NO	0	0	0
	24	L	0	NO	0	NO	0	NO	0	0	0
		R	0	NO	0	NO	0	NO	0	0	0
Solubilised Sulphur Black 1 10 % in Plu	25	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	26	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

NO = Not observable (any erythema is hidden by dark colour of the test item formulation)

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
	23	L	0.19	0.19	0.0	0.19	0.0
Solubilised Sulphur Black 1		R	0.19	0.19	0.0	0.19	0.0
25 % in Plu	24	L	0.20	0.19	-5.0	0.19	-5.0
		R	0.20	0.19	-5.0	0.19	-5.0
	25	L	0.19	0.19	0.0	0.19	0.0
Solubilised Sulphur Black 1		R	0.19	0.19	0.0	0.19	0.0
10 % in Plu	26	L	0.20	0.20	0.0	0.19	-5.0
		R	0.20	0.20	0.0	0.19	-5.0

 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.

Table 7: 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
90	Solubilised Sulphur Black 1	21.7	21.7	0
91	25 %	19.7	19.5	-1
92	in Plu	21.0	20.5	-2
93		22.7	21.4	-6
	Mean	21.3	20.8	-2
	SD	1.3	1.0
94	Solubilised Sulphur Black 1	21.6	21.1	-2
95	10 %	19.3	18.9	-2
96	in Plu	21.0	20.9	0
97		22.4	21.8	-3
	Mean	21.1	20.7	-2
	SD	1.3	1.2
98	Solubilised Sulphur Black 1	19.1	18.9	-1
99	5 %	21.8	21.4	-2
100	in Plu	22.1	20.7	-6
101		21.2	21.5	1
	Mean	21.1	20.6	-2
	SD	1.4	1.2
102	Solubilised Sulphur Black 1	21.7	21.4	-1
103	2.5 %	19.2	18.5	-4
104	in Plu	22.1	21.4	-3
105		21.2	20.6	-3
	Mean	21.1	20.5	-3
	SD	1.3	1.4

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

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

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	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
Solubilised Sulphur Black 1 25 % in Plu	90	N	N	N	N	N	N	N	N	N
	91	N	N	N	N	N	N	N	N	N
	92	N	N	N	N	N	N	N	N	N
	93	N	N	N	N	N	N	N	N	N
Solubilised Sulphur Black 1 10 % in Plu	94	N	N	N	N	N	N	N	N	N
	95	N	N	N	N	N	N	N	N	N
	96	N	N	N	N	N	N	N	N	N
	97	N	N	N	N	N	N	N	N	N
Solubilised Sulphur Black 1 5 % in Plu	98	N	N	N	N	N	N	N	N	N
	99	N	N	N	N	N	N	N	N	N
	100	N	N	N	N	N	N	N	N	N
	101	N	N	N	N	N	N	N	N	N
Solubilised Sulphur Black 1 2.5 % in Plu	102	N	N	N	N	N	N	N	N	N
	103	N	N	N	N	N	N	N	N	N
	104	N	N	N	N	N	N	N	N	N
	105	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)

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	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
Solubilised Sulphur Black 1 25 % in Plu	90	L	0	NO	0	0*	0	0*	0	0	0
		R	0	NO	0	0*	0	0*	0	0	0
	91	L	0	NO	0	0*	0	0*	0	0	0
		R	0	NO	0	0*	0	0*	0	0	0
	92	L	0	NO	0	0*	0	0*	0	0	0
		R	0	NO	0	0*	0	0*	0	0	0
	93	L	0	NO	0	0*	0	0*	0	0	0
		R	0	NO	0	0*	0	0*	0	0	0
Solubilised Sulphur Black 1 10 % in Plu	94	L	0	NO	0	0*	0	0	0	0	0
		R	0	NO	0	0*	0	0	0	0	0
	95	L	0	NO	0	0*	0	0	0	0	0
		R	0	NO	0	0*	0	0	0	0	0
	96	L	0	NO	0	0*	0	0	0	0	0
		R	0	NO	0	0*	0	0	0	0	0
	97	L	0	NO	0	0*	0	0	0	0	0
		R	0	NO	0	0*	0	0	0	0	0
Solubilised Sulphur Black 1 5 % in Plu	98	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	99	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	100	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	101	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Solubilised Sulphur Black 1 2.5 % in Plu	102	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	103	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	104	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	105	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

* Test Item residual is observed on the ears                      NO = Not observable (any erythema is hidden by dark colour of the test item formulation)

Table 10: 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
Solubilised Sulphur Black 1 25 % in Plu	90	N
	91	N
	92	N
	93	N
Solubilised Sulphur Black 1 10 % in Plu	94	N
	95	N
	96	N
	97	N
Solubilised Sulphur Black 1 5 % in Plu	98	N
	99	N
	100	N
	101	N
Solubilised Sulphur Black 1 2.5 % in Plu	102	N
	103	N
	104	N
	105	N

 

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

HCA =a-Hexylcinnamaldehyde

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

N = Normal

Table 11: 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
Solubilised Sulphur Black 1	1776	1751.5	437.9	0.5
25 % in Plu
Solubilised Sulphur Black 1	1676	1651.5	412.9	0.5
10 % in Plu
Solubilised Sulphur Black 1	2308	2283.5	570.9	0.6
5 % in Plu
Solubilised Sulphur Black 1	1879	1854.5	463.6	0.5
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

Interpretation of results:

GHS criteria not met

Conclusions:

In an in vivo local lymphnode assay (LLNA) according to OECD 429, the test item did not show skin sensitising properties.

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 inaqueous 1 % (w/v) Pluronic®PE 9200 (Plu) in the main test. The maximum achievable concentration (based on solubility) was 25 % (w/w). According to the results of the DRF (no adverse effects were observed up to this maximum concentration) the test item was examined in the main test in concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w) as suspension formulations in Plu. Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups (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 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 of above 3) compared to the relevant control (Plu) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 0.5, 0.6 and 0.5 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.63, r = 0.37; evaluated by linear regression using SI values). According to the evaluation, the lack of a significantly increased lymphoproliferation (indicated by an SI of above 3) up to the maximum attainable concentration of 25 % (w/w), based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin-sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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. Based on the results the test item was formulated in inaqueous 1 % (w/v) Pluronic®PE 9200 (Plu) in the main test. The maximum achievable concentration (based on solubility) was 25 % (w/w). According to the results of the DRF (no adverse effects were observed up to this maximum concentration) the test item was examined in the main test in concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w) as suspension formulations in Plu. Appropriate positive control (a-Hexylcinnamaldehyde, HCA), furthermore two negative control groups (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 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 of above 3) compared to the relevant control (Plu) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 0.5, 0.6 and 0.5 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.63, r = 0.37; evaluated by linear regression using SI values). According to the evaluation, the lack of a significantly increased lymphoproliferation (indicated by an SI of above 3) up to the maximum attainable concentration of 25 % (w/w), based on solubility) and also the lack of a significant dose-response relationship are considered evidence that the test item is not a skin-sensitizer.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test item is considered not to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.