Carbonic acid disodium salt, reaction products with aniline, 4-nitrobenzenamine, p-phenylenediamine, sodium sulfide, sulfur and p-toluidine

Endpoint summary

Description of key information

The test item at the maximum feasible concentration (w/v, based on solubility) of 0.1 % was shown to have no skin sensitization potential in the Local Lymph Node Assay.

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:

August 18-October 10, 2016

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:

22th July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test System
Dose Range Finding Animals

Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during
the test: Good conventional
Justification of strain: Same strain is used in the Dose Range Finding test (DRF) and in the main test.
Number of animals: 6 animals (2 animals/groups)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice,12 weeks old (at start of the DRF)

Main Test Animals
Species and strain: CBA/Ca Ola Hsd mice
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)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 9-11 weeks old (at start of the main test)
Body weight range
at starting: 18.6 – 22.7 g
The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
Acclimatization time: 7 days
*Including control animals shared with the concurrent LLNAs. Number of shared animals: 8.

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.
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.

Bedding
Lignocel Hygienic Animal Bedding ) was available to animals during the study.

Identification of Animals

The individual identification of the animals was performed by numbers on the tail. The cages were marked with identification cards, with information (at least) about study code, species, strain, sex, dose group and individual animal numbers.
Randomization
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. 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.

Vehicle:

dimethyl sulphoxide

Concentration:

0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v)

No. of animals per dose:

28 animals; 4 animals/treatment group

Details on study design:

Based on the preliminary test results the maximum attainable concentration (based on solubility) of 0.1 % (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 (0.05 %, 0.025 % and 0.01 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

Animals in the treatment groups were treated with the negative (vehicle) controls (DMSO 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 L 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 L of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 Ci# of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
#Remark: Breaking down of [methyl-3H]-Thymidine in aqueous solution (about 3 % per month) was taken into account as necessary when 3HTdR solution was prepared.

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 betha-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The betha-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.

Positive control substance(s):

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

Statistics:

Randomization based on body weigt was checked by SPSS PC+
Calculation of stimulation indices were made by EXCEL software

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.
Significant lymphoproliferative response (SI > 3) was noted for HCA (SI = 13.8). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

SI at all test concentrations below 3

Parameter:

SI

Value:

1.9

Test group / Remarks:

0.01%

Parameter:

SI

Value:

1.6

Test group / Remarks:

0.025%

Parameter:

SI

Value:

2.4

Test group / Remarks:

0.05%

Parameter:

SI

Value:

2.5

Test group / Remarks:

0.1%

Cellular proliferation data / Observations:

No significant, treatment related effect on body weights was observed during the test. Although body weights decreased by 7 % were observed in the groups treated with DMSO or 0.01 % (w/v) formulation of the test item (1/4 animals in both) the effect was considered neither significant nor treatment related.
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.
No significant lymphoproliferative response compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.5, 2.4, 1.6 and 1.9 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively.
Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.19, r = 0.81).

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 (%)
290	test item	21.5	20.5	-5
291	0.1 % in DMSO	20.1	20.2	0
	Mean	20.8	20.4	-2
292	test item	21.5	20.4	-5
293	0.05 % in DMSO	20.1	19.8	-1
	Mean	20.8	20.1	-3
294	test item	21.7	20.7	-5
295	0.025 % in DMSO	20.0	19.2	-4
	Mean	20.9	20.0	-4

 

*Terminal body weights were measured on Day 6.

DMSO = Dimethyl sulfoxide

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
Yellow 22 0.1 % in DMSO	290	N	N	N	N	N	N	N	N	N
	291	N	N	N	N	N	N	N	N	N
Yellow 22 0.05 % in DMSO	292	N	N	N	N	N	N	N	N	N
	293	N	N	N	N	N	N	N	N	N
Yellow 22 0.025 % in DMSO	294	N	N	N	N	N	N	N	N	N
	295	N	N	N	N	N	N	N	N	N

PT = Prior to the treatment

AT = After the treatment

DMSO = Dimethyl sulfoxide

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
test item 0.1 % in DMSO	290	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	291	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
test item 0.05 % in DMSO	292	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	293	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
test item 0.025 % in DMSO	294	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	295	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

DMSO = Dimethyl sulfoxide

 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
	290	L	0.21	0.22	4.8	0.24	14.3
test item		R	0.21	0.21	0.0	0.24	14.3
0.1 % in DMSO	291	L	0.21	0.21	0.0	0.24	14.3
		R	0.21	0.21	0.0	0.25	19.0
	292	L	0.21	0.21	0.0	0.24	14.3
test item		R	0.21	0.21	0.0	0.23	9.5
0.05 % in DMSO	293	L	0.20	0.21	5.0	0.23	15.0
		R	0.20	0.20	0.0	0.23	15.0
	294	L	0.21	0.21	0.0	0.22	4.8
test item		R	0.21	0.21	0.0	0.23	9.5
0.025 % in DMSO	295	L	0.20	0.20	0.0	0.24	20.0
		R	0.20	0.20	0.0	0.24	20.0

L = Left

R = Right

DMSO = Dimethyl sulfoxide

 

* 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)	(%)
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
465	Vehicle control for the test item:	20.7	19.3	-7
496	DMSO	22.3	21.5	-4
497		19.1	20.1	5
538		20.5	20.9	2
	Mean	20.7	20.5	-1
	SD	1.3	1.0
466	test item	20.0	20.9	5
498	0.1 %	22.7	23.4	3
499	in DMSO	19.2	20.6	7
539		20.7	21.4	3
	Mean	20.7	21.6	4
	SD	1.5	1.3
467	test item	20.3	19.9	-2
500	0.05 %	19.4	20.2	4
501	in DMSO	21.4	20.9	-2
540		22.2	22.6	2
	Mean	20.8	20.9	0
	SD	1.2	1.2
502	test item	20.7	20.9	1
503	0.025 %	22.4	23.2	4
541	in DMSO	19.4	20.8	7
542		20.2	21.7	7
	Mean	20.7	21.7	5
	SD	1.3	1.1
504	test item	22.5	21.0	-7
505	0.01 %	19.8	19.9	1
506	in DMSO	20.6	22.2	8
543		19.2	19.7	3
	Mean	20.5	20.7	1
	SD	1.4	1.2

 

HCA =a-Hexylcinnamaldehyde

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

DMSO = Dimethyl sulfoxide

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: DMSO	465	N	N	N	N	N	N	N	N	N
	496	N	N	N	N	N	N	N	N	N
	497	N	N	N	N	N	N	N	N	N
	538	N	N	N	N	N	N	N	N	N
test item 0.1 % in DMSO	466	N	N	N	N	N	N	N	N	N
	498	N	N	N	N	N	N	N	N	N
	499	N	N	N	N	N	N	N	N	N
	539	N	N	N	N	N	N	N	N	N
test item 0.05 % in DMSO	467	N	N	N	N	N	N	N	N	N
	500	N	N	N	N	N	N	N	N	N
	501	N	N	N	N	N	N	N	N	N
	540	N	N	N	N	N	N	N	N	N
test item 0.025 % in DMSO	502	N	N	N	N	N	N	N	N	N
	503	N	N	N	N	N	N	N	N	N
	541	N	N	N	N	N	N	N	N	N
	542	N	N	N	N	N	N	N	N	N
test item 0.01 % in DMSO	504	N	N	N	N	N	N	N	N	N
	505	N	N	N	N	N	N	N	N	N
	506	N	N	N	N	N	N	N	N	N
	543	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)

DMSO = Dimethyl sulfoxide

 

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	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: DMSO	465	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	496	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	497	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	538	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

DMSO = Dimethyl sulfoxide

HCA =a-Hexylcinnamaldehyde

Erythema Scores in the Main Test

Dose Group	Animal Number	Ears	Days
			1		2		3		4	5	6
			PT	AT	PT	AT	PT	AT
test item 0.1 % in DMSO	466	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	498	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	499	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	539	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
test item 0.05 % in DMSO	467	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	500	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	501	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	540	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
test item 0.025 % in DMSO	502	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	503	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	541	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	542	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
test item 0.01 % in DMSO	504	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	505	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	506	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	543	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

DMSO = Dimethyl sulfoxide

 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: DMSO	465	N
	496	N
	497	N
	538	N
test item 0.1 % in DMSO	466	N
	498	N
	499	N
	539	N
test item 0.05 % in DMSO	467	N
	500	N
	501	N
	540	N
test item 0.025 % in DMSO	502	N
	503	N
	541	N
	542	N
test item 0.01 % in DMSO	504	N
	505	N
	506	N
	543	N

 

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

HCA =a-Hexylcinnamaldehyde

DMSO = Dimethyl sulfoxide

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:	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:	2632	2590.5	647.6	1.0
DMSO
test item	6447	6405.5	1601.4	2.5
0.1 % in DMSO
test item	6192	6150.5	1537.6	2.4
0.05 % in DMSO
test item	4184	4142.5	1035.6	1.6
0.025 % in DMSO
test item	4939	4897.5	1224.4	1.9
0.01 % in DMSO

 

HCA =a-Hexylcinnamaldehyde

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

DMSO = Dimethyl sulfoxide

 Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 41.5

# Number of animals/group = 4

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

The Local Lymph Node Assay was conducted to evaluate the skin sensitization potential of the test item following dermal exposure. According to results of the DRF (where no adverse effect was observed up to the maximum achievable concentration of 0.1 % (w/v)) the test item was examined in the main test as 0.1 %, 0.05 %, 0.025 % or 0.01 % (w/v) formulations in DMSO. 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 over the relevant control (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 (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.5, 2.4, 1.6 and 1.9 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.19, r = 0.81; 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 0.1 % (w/v, 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. In conclusion, under the conditions of the present assay,the test item tested at the maximum feasible concentration of 0.1 % (w/v, based on solubility) and also at concentrations of 0.05 %, 0.025 % or 0.01 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMSO) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The Local Lymph Node Assay was conducted to evaluate the skin sensitization potential of the test item following dermal exposure. According to results of the DRF (where no adverse effect was observed up to the maximum achievable concentration of 0.1 % (w/v)) the test item was examined in the main test as 0.1 %, 0.05 %, 0.025 % or 0.01 % (w/v) formulations in DMSO. 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 over the relevant control (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 (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 2.5, 2.4, 1.6 and 1.9 at test item concentrations of 0.1 %, 0.05 %, 0.025 % and 0.01 % (w/v), respectively. 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 0.1 % (w/v, 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. In conclusion, under the conditions of the present assay, the test item tested up to the maximum feasible concentration of 0.1 % (w/v, based on solubility) was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

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 (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.