Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2017 - 15 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

EC Number:

288-914-8

EC Name:

Phenol, 4-(9H-carbazol-3-ylamino)-, reaction products with sodium sulfide (Na2(Sx)) and sulfur, leuco deriv.

Cas Number:

85940-25-6

Molecular formula:

Molecular formula is not available

IUPAC Name:

Reaction product of phenol, 4-​(9H-​carbazol-​3-​ylamino)​- with sodium polysulfide, leuco derivatives

Test material form:

solid: particulate/powder

Details on test material:

Test Item: Leuco Sulfur Blue 20P
Appearance: Black powder

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, 12 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 12 weeks old (at start of the main test)
Body weight rangeat starting: 18.6 – 22.8 g .The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
Acclimatization time: 14 days

Husbandry
Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing duringacclimatization 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 %
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.

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.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

test item concentrations: 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w)

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.

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.

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.

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).

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.

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.
 
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 % (w/v) 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 stored at 2-8 °C. On the day of measurement samples were dispersed again using (an ultrasonic water bath), 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

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 when compared to the concurrent control (SI = 10.0), thus confirming the validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

SI values were below 3 at all test concentrations

Results 2

Parameter:

SI

Value:

0.5

Variability:

p = 0.78, r = 0.22

Test group / Remarks:

at test item concentrations of 12.5 % (w/w)

Results 3

Parameter:

SI

Value:

1.3

Variability:

p = 0.78, r = 0.22

Test group / Remarks:

at test item concentrations of 6.25 % (w/w)

Results 4

Parameter:

SI

Value:

0.4

Variability:

p = 0.78, r = 0.22

Test group / Remarks:

at test item concentrations of 3.13 % (w/w)

Results 5

Parameter:

SI

Value:

0.3

Variability:

p = 0.78, r = 0.22

Test group / Remarks:

at test item concentrations o1.56 % (w/w)

Cellular proliferation data / Observations:

No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control [50 % (v/v) DMSO in water] was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 1.3, 0.4 and 0.3 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.78, r = 0.22; 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 12.5 % (w/w) as well as the lack of a significant dose-response relationship is considered as evidence that Leuco Sulfur Blue 20P is not a skin sensitizer.
No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.

Any other information on results incl. tables

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)	(%)
45	Vehicle control	20.0	20.0	0
46	for the positive control:	22.3	22.1	-1
47	AOO	19.1	19.5	2
48		21.6	21.7	0
	Mean	20.8	20.8	0
	SD	1.5	1.3
49	Positive control:	20.3	20.4	0
50	25 % HCA	21.5	20.3	-6
51	in AOO	22.8	22.1	-3
52		19.3	19.8	3
	Mean	21.0	20.7	-2
	SD	1.5	1.0
53	Vehicle control	21.0	21.5	2
54	for the test item:	21.4	22.0	3
55	50 % DMSO in water	22.0	22.5	2
56		18.6	18.9	2
	Mean	20.8	21.2	2
	SD	1.5	1.6
89	Leuco Sulfur Blue 20P	20.0	20.2	1
90	12.5 %	22.7	20.7	-9
91	in DMSO : water	21.7	20.3	-6
92		19.7	20.4	4
	Mean	21.0	20.4	-3
	SD	1.4	0.2
93	Leuco Sulfur Blue 20P	19.8	20.4	3
94	6.25 %	19.3	19.7	2
95	in DMSO : water	21.7	21.0	-3
96		22.3	20.8	-7
	Mean	20.8	20.5	-1
	SD	1.5	0.6
97	Leuco Sulfur Blue 20P	19.0	19.3	2
98	3.13 %	22.1	20.0	-10
99	in DMSO : water	19.8	20.0	1
100		21.9	20.8	-5
	Mean	20.7	20.0	-3
	SD	1.5	0.6
101	Leuco Sulfur Blue 20P	19.5	20.0	3
102	1.56 %	22.1	20.2	-9
103	in DMSO : water	22.0	24.7	12
104		19.8	19.7	-1
	Mean	20.9	21.2	1
	SD	1.4	2.4

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

DMSO = Dimethyl sulfoxide                                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	45	N	N	N	N	N	N	N	N	N
	46	N	N	N	N	N	N	N	N	N
	47	N	N	N	N	N	N	N	N	N
	48	N	N	N	N	N	N	N	N	N
Positive control: 25 % HCA in AOO	49	N	N	N	N	N	N	N	N	N
	50	N	N	N	N	N	N	N	N	N
	51	N	N	N	N	N	N	N	N	N
	52	N	N	N	N	N	N	N	N	N
Vehicle control for the test item: 50 % DMSO in water	53	N	N	N	N	N	N	N	N	N
	54	N	N	N	N	N	N	N	N	N
	55	N	N	N	N	N	N	N	N	N
	56	N	N	N	N	N	N	N	N	N
Leuco Sulfur Blue 20P 12.5 % in DMSO : water	89	N	N	N	N	N	N	N	N	N
	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
Leuco Sulfur Blue 20P 6.25 % in DMSO : water	93	N	N	N	N	N	N	N	N	N
	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
Leuco Sulfur Blue 20P 3.13 % in DMSO : water	97	N	N	N	N	N	N	N	N	N
	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
Leuco Sulfur Blue 20P 1.56 % in DMSO : water	101	N	N	N	N	N	N	N	N	N
	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

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	45	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	46	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	47	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	48	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	49	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	50	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	51	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	52	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: 50 % DMSO in water	53	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	54	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	55	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	56	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Leuco Sulfur Blue 20P 12.5 % in DMSO : water	89	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	90	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	91	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	92	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Leuco Sulfur Blue 20P 6.25 % in DMSO : water	93	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	94	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	95	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	96	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Leuco Sulfur Blue 20P 3.13 % in DMSO : water	97	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	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
Leuco Sulfur Blue 20P 1.56 % in DMSO : water	101	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	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

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

AOO = Acetone: Olive oil 4:1 (v/v) mixture                    HCA =a-Hexylcinnamaldehyde                  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	45	N
	46	N
	47	N
	48	N
Positive control: 25 % HCA in AOO	49	Larger than the relevant control (AOO)
	50	Larger than the relevant control (AOO)
	51	Larger than the relevant control (AOO)
	52	Larger than the relevant control (AOO)
Vehicle control for the test item: 50 % DMSO in water	53	N
	54	N
	55	N
	56	N
Leuco Sulfur Blue 20P 12.5 % in DMSO : water	89	N
	90	N
	91	N
	92	N
Leuco Sulfur Blue 20P 6.25 % in DMSO : water	93	N
	94	N
	95	N
	96	N
Leuco Sulfur Blue 20P 3.13 % in DMSO : water	97	N
	98	N
	99	N
	100	N
Leuco Sulfur Blue 20P 1.56 % in DMSO : water	101	N
	102	N
	103	N
	104	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:	9821	9798.5	2449.6	1.0
AOO
Positive control:	97574	97551.5	24387.9	10.0
25 % HCA in AOO
Vehicle control for the test item:	11911	11888.5	2972.1	1.0
50 % DMSO in water
Leuco Sulfur Blue 20P	6516	6493.5	1623.4	0.5
12.5 % in DMSO : water
Leuco Sulfur Blue 20P	15024	15001.5	3750.4	1.3
6.25 % in DMSO : water
Leuco Sulfur Blue 20P	4728	4705.5	1176.4	0.4
3.13 % in DMSO : water
Leuco Sulfur Blue 20P	3671	3648.5	912.1	0.3
1.56 % in DMSO : water

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= 22.5

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined not to have a skin sensitization potential.

Executive summary:

The aim of this study according to OECD 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 according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. In general, very low solubility of the test item was observed. The test item was formulated in water resulting in a thick suspension (slurry) at a nominal concentration of 25 % (w/w) prior to dilution with an appropriate vehicle (DMSO) in a ratio of 1:1 (v/v). The 12.5 % (w/w) formulation prepared in this way was an adequately stable and homogeneous suspension for application on the dorsum of ears of animals. No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test (12.5 % and 6.25 % (w/w) were tested). According to this the test item was examined in the main test at concentrations of 12.5 % and at 6.25 %, 3.13 % or 1.56 % (w/w; prepared by serial dilution in DMSO : water 1:1 (v/v) mixture) as suspension formulations. An appropriate positive control (a-Hexylcinnamaldehyde, HCA) and furthermore two negative control groups dosed withDMSO : water 1:1 (v/v) mixture(as vehicle control for the test groups) or AOO (as vehicle control for the positive control group) were employed.

The positive control item (25 % (w/v) HCA in Acetone : Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation when compared to the concurrent control (SI = 10.0), thus confirming the validity of the assay. No mortality or signs of systemic toxicity were observed during the test. No significant treatment related effect on the body weights was observed in any dose group. 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 [50 % (v/v) DMSO in water] was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.5, 1.3, 0.4 and 0.3 at test item concentrations of 12.5 %, 6.25 %, 3.13 % and 1.56 % (w/w), respectively. No significant dose-response relationship was observed (p = 0.78, r = 0.22; 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 12.5 % (w/w) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitizer.