1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 23 - April 28, 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:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May, 2008

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:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

EC Number:

309-269-1

EC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Cas Number:

100208-66-0

Molecular formula:

not applicable

IUPAC Name:

1,3-Benzenediamine, 4-methyl-, reaction products with 4-nitrobenzenamine, p-phenylenediamine and sodium sulfide (Na2(Sx))

Test material form:

solid: particulate/powder

Details on test material:

Test item: Brown 3
Appearance: black powder
CAS No. 100208-66-0
EC No. 309-269-1
Storage: room temperature

Specific details on test material used for the study:

Date of production: 16.06.2015
Expiration date: 16.06.2020

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:

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; 11-12 weeks old (at start of the main test)
Body weight range at starting: 17.4 – 21.5 g
The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
Acclimatization time: 7 days

Husbandry
Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
The same conditions were used for the dose range finding and main test animals. There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.
Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice.

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. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.
 
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.

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.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

test item concentrations: 12.5%, 6.25%, 3.13%, 1.56%

No. of animals per dose:

4 animals/treatment group

Details on study design:

Based on the preliminary test results, 12.5 % (w/w) was used as the maximum attainable concentration in the main test with the aim of testing the highest possible concentration and formulated according to the procedure recommended by the Sponsor. The test item was tested also at three additional lower concentrations (6.25 %, 3.13 % and 1.56 %, w/w) to evaluate a 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 controls (vehicles), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.

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

Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test. All animals treated were processed and therefore no treatment group was excluded from the evaluation.
Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 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. Then 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 stored at room temperature protected from light until measurement in the -scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The -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 = 6.6), thus confirming the validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

SI at all test concentrations below 3

Results 2

Parameter:

SI

Value:

2.4

Variability:

p=0.20; r= 0.80

Test group / Remarks:

at test item concentration of 12.5%

Results 3

Parameter:

SI

Value:

1

Variability:

p=0.20; r= 0.80

Test group / Remarks:

at test item concentration of 6.25%

Results 4

Parameter:

SI

Value:

1.3

Variability:

p=0.20; r= 0.80

Test group / Remarks:

at test item concentration of 3.13%

Results 5

Parameter:

SI

Value:

1.3

Variability:

p=0.20; r= 0.80

Test group / Remarks:

at test item concentration of 1.56%

Cellular proliferation data / Observations:

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 Brown 3 at the applied test concentrations. The observed stimulation index values were 2.4, 1.0, 1.3 and 1.3 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 = 0.20, r = 0.80; 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 Brown 3 is not a skin sensitizer.

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)	(%)
308	Vehicle control	20.2	21.2	5
309	for the positive control:	18.2	18.2	0
310	AOO	20.5	19.3	-6
345		21.5	22.4	4
	Mean	20.1	20.3	1
	SD	1.4	1.9
311	Positive control:	18.1	17.4	-4
312	25 % HCA	20.8	20.1	-3
313	in AOO	20.0	20.8	4
346		21.3	21.3	0
	Mean	20.1	19.9	-1
	SD	1.4	1.7
314	Vehicle control	19.8	19.6	-1
315	for the test item:	20.9	20.4	-2
316	50 % (v/v) DMSO in water	17.9	18.2	2
347		21.0	20.9	0
	Mean	19.9	19.8	-1
	SD	1.4	1.2
317	Brown 3	17.6	17.7	1
318	12.5 %	21.1	21.8	3
319	in DMSO : water	19.3	19.1	-1
348		20.7	20.0	-3
	Mean	19.7	19.7	0
	SD	1.6	1.7
320	Brown 3	19.5	19.3	-1
321	6.25 %	17.6	18.1	3
322	in DMSO : water	21.1	21.4	1
349		20.9	20.3	-3
	Mean	19.8	19.8	0
	SD	1.6	1.4
323	Brown 3	17.4	17.4	0
324	3.13 %	19.4	19.6	1
350	in DMSO : water	21.1	19.8	-6
351		20.6	20.5	0
	Mean	19.6	19.3	-2
	SD	1.6	1.3
325	Brown 3	21.4	20.7	-3
326	1.56 %	19.9	19.0	-5
327	in DMSO : water	18.0	18.1	1
352		20.5	21.4	4
	Mean	20.0	19.8	-1
	SD	1.4	1.5

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	308	N	N	N	N	N	N	N	N	N
	309	N	N	N	N	N	N	N	N	N
	310	N	N	N	N	N	N	N	N	N
	345	N	N	N	N	N	N	N	N	N
Positive control: 25 % HCA in AOO	311	N	N	N	N	N	N	N	N	N
	312	N	N	N	N	N	N	N	N	N
	313	N	N	N	N	N	N	N	N	N
	346	N	N	N	N	N	N	N	N	N
Vehicle control for the test item: 50 % (v/v) DMSO in water	314	N	N	N	N	N	N	N	N	N
	315	N	N	N	N	N	N	N	N	N
	316	N	N	N	N	N	N	N	N	N
	347	N	N	N	N	N	N	N	N	N
Brown 3 12.5 % in DMSO : water	317	N	N	N	N	N	N	N	N	N
	318	N	N	N	N	N	N	N	N	N
	319	N	N	N	N	N	N	N	N	N
	348	N	N	N	N	N	N	N	N	N
Brown 3 6.25 % in DMSO : water	320	N	N	N	N	N	N	N	N	N
	321	N	N	N	N	N	N	N	N	N
	322	N	N	N	N	N	N	N	N	N
	349	N	N	N	N	N	N	N	N	N
Brown 3 3.13 % in DMSO : water	323	N	N	N	N	N	N	N	N	N
	324	N	N	N	N	N	N	N	N	N
	350	N	N	N	N	N	N	N	N	N
	351	N	N	N	N	N	N	N	N	N
Brown 3 1.56 % in DMSO : water	325	N	N	N	N	N	N	N	N	N
	326	N	N	N	N	N	N	N	N	N
	327	N	N	N	N	N	N	N	N	N
	352	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	308	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	309	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	310	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	345	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	311	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	312	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	313	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	346	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 % (v/v) DMSO in water	314	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	315	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	316	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	347	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Brown 3 12.5 % in DMSO : water	317	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	318	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	319	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	348	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Brown 3 6.25 % in DMSO : water	320	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	321	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	322	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	349	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Brown 3 3.13 % in DMSO : water	323	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	324	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	350	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	351	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Brown 3 1.56 % in DMSO : water	325	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	326	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	327	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	352	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	308	N
	309	N
	310	N
	345	N
Positive control: 25 % HCA in AOO	311	Larger than the relevant control (AOO)
	312	Larger than the relevant control (AOO)
	313	Larger than the relevant control (AOO)
	346	Larger than the relevant control (AOO)
Vehicle control for the test item: 50 % (v/v) DMSO in water	314	N
	315	N
	316	N
	347	N
Brown 3 12.5 % in DMSO : water	317	N
	318	N
	319	N
	348	N
Brown 3 6.25 % in DMSO : water	320	N
	321	N
	322	N
	349	N
Brown 3 3.13 % in DMSO : water	323	N
	324	N
	350	N
	351	N
Brown 3 1.56 % in DMSO : water	325	N
	326	N
	327	N
	352	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:	3007	2985.0	746.3	1.0
AOO
Positive control:	19794	19772.0	4943.0	6.6
25 % HCA in AOO
Vehicle control for the test item:	1719	1697.0	424.3	1.0
50 % (v/v) DMSO in water
Brown 3	4081	4059.0	1014.8	2.4
12.5 % in DMSO : water
Brown 3	1760	1738.0	434.5	1.0
6.25 % in DMSO : water
Brown 3	2261	2239.0	559.8	1.3
3.13 % in DMSO : water
Brown 3	2231	2209.0	552.3	1.3
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

# Number of animals/group = 4

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

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. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. The maximum achievable soluble concentration was 0.05 % (w/v) in DMSO using ultrasonic dispersion and stirring. No adequate homogeneous formulation (solution or suspension) was obtained with any of the vehicles used in the solvent trial above this concentration. To find an appropriate formulation at higher concentration 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; see section 6.1.3) according to the Sponsor’s request. 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 up to 12.5 % (w/w) concentration. According to this the test item was examined in the main test at concentration 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 with DMSO : 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 = 6.6), 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 2.4, 1.0, 1.3 and 1.3 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 = 0.20, r = 0.80; 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 substance is not a skin sensitizer.