Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2018 - 20 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

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:

Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

EC Number:

276-059-3

EC Name:

Formamide, N,N'-1,4-phenylenebis-, reaction products with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Cas Number:

71838-68-1

Molecular formula:

not applicable

IUPAC Name:

Reaction product of formamide, N,N'-1,4-phenylenebis- with 4-methyl-1,3-benzenediamine and sulfur, leuco derivatives

Test material form:

other: presscake

Remarks:

press cake

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:

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 duringthe 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; 12 weeks old (at start of the main test)
Body weight range at starting: 21.3 – 23.6 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.
There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phase. 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. 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.

Bedding

Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg (Germany) Holzmühle 1) 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.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

50 %, 25 %, 10 % and 5 % (w/v)

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.

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 (see Table 2) 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 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 (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. 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

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.

Signs of Irritation

Irritation was monitored by erythema scoring during according to criteria depicted in Table 3 during the whole test (Day 1 to Day 6). Ear thickness was measured in all treatment groups. Ear thickness measurement was taken by using a digital micrometer on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Individual records were maintained for both observations.

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 an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item.

 
Interpretation of the Results

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.

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 = 7.9), thus confirming the validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

The SI values at 25%, 10% and 5% were below 3. The SI at 50% was considered as not relevant.

Results 2

Parameter:

SI

Value:

4.4

Variability:

p = 0.04, r = 0.96

Test group / Remarks:

at test item concentrations of 50 %

Remarks on result:

other: The increased ear thickness values observed in the 50 % (w/v) dose group may indicate a mild irritation.

Results 3

Parameter:

SI

Value:

2.5

Variability:

p = 0.04, r = 0.96

Test group / Remarks:

at test item concentration of 25 %,

Results 4

Parameter:

SI

Value:

2.3

Variability:

p = 0.04, r = 0.96

Test group / Remarks:

at test item concentrations of 10 %

Results 5

Parameter:

SI

Value:

2.1

Variability:

p = 0.04, r = 0.96

Test group / Remarks:

at test item concentrations of 5 %

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. As a sign of a possible irritation significantly increased ear thicknesses (i.e. > 25 % increase compared to the initial values) were observed in the 50 % (w/v) dose group. Ear thicknesses in the other treatment groups did not differ significantly from the initial values or the values measured in the relevant control group during the test. No erythema or other local effects were observed in any treatment group.

A significant lymphoproliferative response (SI >= 3) compared to the concurrent control (50 % DMSO) was noted for the test item presscake at a concentration of 50 % (w/v). No significant lymphoproliferative response was observed at the other test concentrations. The observed stimulation index values were 4.4, 2.5, 2.3 and 2.1 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. A statistically significant linear dose-response correlation was observed (p = 0.04, r = 0.96, evaluated by linear regression using the SI values). The increased ear thickness values observed in the 50 % (w/v) dose group may indicate a mild irritation. It is also considered that although 50 % (v/v) DMSO in water was deemed to be the most appropriate negative control for all test item formulation proportion of DMSO could be slightly higher in the 50 % (w/v) test item formulation. As DMSO itself usually results in higher proliferation background its higher proportion could result in overestimation of the SI value in this dose group. According to this the observed SI value of 4.4 is considered as a consequence of an effect other than real sensitization. The stimulation index values of 2.5, 2.3 and 2.1 observed at 25 %, 10 % and 5 % (w/v) concentrations (respectively) did not indicate sensitization potential of the test item.

Any other information on results incl. tables

Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes

Animal	Dose Group	Initial	Terminal	Body Weight
Number		Body Weight	Body Weight	Change
		(g)	(g)	(%)
200	Vehicle control	21.6	20.9	-3
201	for the positive control:	21.3	20.4	-4
202	AOO	23.4	22.9	-2
203		22.6	21.7	-4
	Mean	22.2	21.5	-3
	SD	1.0	1.1
204	Positive control:	21.7	22.1	2
205	25 % HCA	21.3	21.1	-1
206	in AOO	23.5	23.4	0
207		22.7	22.8	0
	Mean	22.3	22.4	0
	SD	1.0	1.0
208	Vehicle control for the test item:	23.6	23.8	1
209	50 % (v/v) DMSO in water	21.3	21.7	2
210		21.6	22.4	4
211		22.7	23.5	4
	Mean	22.3	22.9	2
	SD	1.1	1.0
212	Leuco Sulfur Brown 26 presscake	21.8	22.4	3
213	50 %	23.3	21.6	-7
214	in DMSO	21.3	21.2	0
215		22.7	24.0	6
	Mean	22.3	22.3	0
	SD	0.9	1.2
216	Leuco Sulfur Brown 26 presscake	21.7	20.5	-6
217	25 %	23.5	22.8	-3
218	in DMSO:water	21.3	21.1	-1
219		22.7	22.0	-3
	Mean	22.3	21.6	-3
	SD	1.0	1.0
220	Leuco Sulfur Brown 26 presscake	22.9	22.8	0
221	10 %	21.5	21.3	-1
222	in DMSO:water	21.7	22.1	2
223		23.4	23.1	-1
	Mean	22.4	22.3	0
	SD	0.9	0.8
224	Leuco Sulfur Brown 26 presscake	22.9	21.7	-5
225	5 %	23.3	23.5	1
226	in DMSO:water	21.5	22.7	6
227		21.8	22.8	5
	Mean	22.4	22.7	1
	SD	0.9	0.7

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

DMSO = Dimethyl sulfoxide                               SD = Standard Deviation

Clinical Observations

Dose Group	Animal Number	Days
		1		2		3		4	5	6
		PT	AT	PT	AT	PT	AT
Vehicle control for the positive control: AOO	200	N	N	N	N	N	N	N	N	N
	201	N	N	N	N	N	N	N	N	N
	202	N	N	N	N	N	N	N	N	N
	203	N	N	N	N	N	N	N	N	N
Positive control: 25 % HCA in AOO	204	N	N	N	N	N	N	N	N	N
	205	N	N	N	N	N	N	N	N	N
	206	N	N	N	N	N	N	N	N	N
	207	N	N	N	N	N	N	N	N	N
Vehicle control for the test item: 50 % (v/v) DMSO in water	208	N	N	N	N	N	N	N	N	N
	209	N	N	N	N	N	N	N	N	N
	210	N	N	N	N	N	N	N	N	N
	211	N	N	N	N	N	N	N	N	N
Leuco Sulfur Brown 26 presscake 50 % in DMSO	212	N	N	N	N	N	N	N	N	N
	213	N	N	N	N	N	N	N	N	N
	214	N	N	N	N	N	N	N	N	N
	215	N	N	N	N	N	N	N	N	N
Leuco Sulfur Brown 26 presscake 25 % in DMSO:water	216	N	N	N	N	N	N	N	N	N
	217	N	N	N	N	N	N	N	N	N
	218	N	N	N	N	N	N	N	N	N
	219	N	N	N	N	N	N	N	N	N
Leuco Sulfur Brown 26 presscake 10 % in DMSO:water	220	N	N	N	N	N	N	N	N	N
	221	N	N	N	N	N	N	N	N	N
	222	N	N	N	N	N	N	N	N	N
	223	N	N	N	N	N	N	N	N	N
Leuco Sulfur Brown 26 presscake 5 % in DMSO:water	224	N	N	N	N	N	N	N	N	N
	225	N	N	N	N	N	N	N	N	N
	226	N	N	N	N	N	N	N	N	N
	227	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

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	200	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	201	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	202	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	203	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	204	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	205	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	206	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	207	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	208	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	209	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	210	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	211	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Leuco Sulfur Brown 26 presscake 50 % in DMSO	212	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	213	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	214	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	215	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
Leuco Sulfur Brown 26 presscake 25 % in DMSO:water	216	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	217	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	218	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
	219	L	0	0*	0	0*	0	0*	0	0	0
		R	0	0*	0	0*	0	0*	0	0	0
Leuco Sulfur Brown 26 presscake 10 % in DMSO:water	220	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	221	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	222	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	223	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
Leuco Sulfur Brown 26 presscake 5 % in DMSO:water	224	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	225	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	226	L	0	0	0	0	0	0	0	0	0
		R	0	0	0	0	0	0	0	0	0
	227	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

* Test Item residual is observed on the ears                      

Individual Ear Thickness Values and the Deviations from the Initial Values

Dose Group	Animal	Ears	Day 1^*	Day 3^$	Day 3	Day 6^#	Day 6
	Number		value (mm)	value (mm)	% deviation	value (mm)	% deviation
	200	L	0.19	0.20	5.3	0.19	0.0
		R	0.19	0.20	5.3	0.19	0.0
Vehicle control for the positive control:	201	L	0.19	0.20	5.3	0.19	0.0
AOO		R	0.19	0.20	5.3	0.19	0.0
	202	L	0.20	0.19	-5.0	0.20	0.0
		R	0.20	0.19	-5.0	0.20	0.0
	203	L	0.20	0.19	-5.0	0.19	-5.0
		R	0.20	0.19	-5.0	0.19	-5.0
	204	L	0.20	0.20	0.0	0.22	10.0
		R	0.20	0.20	0.0	0.22	10.0
Positive control:	205	L	0.20	0.20	0.0	0.19	-5.0
25 % HCA in AOO		R	0.20	0.20	0.0	0.19	-5.0
	206	L	0.19	0.20	5.3	0.23	21.1
		R	0.19	0.20	5.3	0.23	21.1
	207	L	0.20	0.20	0.0	0.20	0.0
		R	0.20	0.20	0.0	0.20	0.0
	208	L	0.18	0.20	11.1	0.20	11.1
		R	0.18	0.20	11.1	0.20	11.1
Vehicle control for the test item:	209	L	0.19	0.20	5.3	0.19	0.0
50 % (v/v) DMSO in water		R	0.19	0.20	5.3	0.19	0.0
	210	L	0.19	0.20	5.3	0.19	0.0
		R	0.19	0.20	5.3	0.19	0.0
	211	L	0.19	0.20	5.3	0.20	5.3
		R	0.19	0.20	5.3	0.20	5.3

L = Left

R = Right

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

HCA =a-Hexylcinnamaldehyde

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 Ear Thickness Values and the Deviations from the Initial Values

Dose Group	Animal	Ears	Day 1^*	Day 3^$	Day 3	Day 6^#	Day 6
	Number		value (mm)	value (mm)	% deviation	value (mm)	% deviation
	212	L	0.20	0.23	15.0	0.25	25.0
		R	0.20	0.23	15.0	0.23	15.0
Leuco Sulfur Brown 26 presscake	213	L	0.19	0.19	0.0	0.25	31.6
50 % in DMSO		R	0.19	0.19	0.0	0.25	31.6
	214	L	0.19	0.19	0.0	0.22	15.8
		R	0.19	0.19	0.0	0.24	26.3
	215	L	0.19	0.24	26.3	0.23	21.1
		R	0.19	0.24	26.3	0.25	31.6
	216	L	0.19	0.21	10.5	0.23	21.1
		R	0.19	0.21	10.5	0.23	21.1
Leuco Sulfur Brown 26 presscake	217	L	0.19	0.20	5.3	0.21	10.5
25% in DMSO:water		R	0.19	0.20	5.3	0.23	21.1
	218	L	0.19	0.20	5.3	0.23	21.1
		R	0.19	0.20	5.3	0.22	15.8
	219	L	0.19	0.20	5.3	0.22	15.8
		R	0.19	0.20	5.3	0.22	15.8
	220	L	0.20	0.20	0.0	0.21	5.0
		R	0.20	0.20	0.0	0.21	5.0
Leuco Sulfur Brown 26 presscake	221	L	0.19	0.20	5.3	0.21	10.5
10 % in DMSO:water		R	0.19	0.20	5.3	0.20	5.3
	222	L	0.20	0.20	0.0	0.21	5.0
		R	0.20	0.20	0.0	0.21	5.0
	223	L	0.18	0.20	11.1	0.21	16.7
		R	0.18	0.20	11.1	0.21	16.7
	224	L	0.20	0.19	-5.0	0.21	5.0
		R	0.20	0.19	-5.0	0.21	5.0
Leuco Sulfur Brown 26 presscake	225	L	0.20	0.20	0.0	0.19	-5.0
5 % in DMSO:water		R	0.20	0.20	0.0	0.21	5.0
	226	L	0.19	0.19	0.0	0.20	5.3
		R	0.19	0.19	0.0	0.20	5.3
	227	L	0.19	0.19	0.0	0.20	5.3
	212	L	0.20	0.23	15.0	0.25	25.0

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.

  Visual Observations of the Lymph Nodes

Dose Group	Animal Number	Appearance of Lymph Nodes
Vehicle control for the positive control: AOO	200	N
	201	N
	202	N
	203	N
Positive control: 25 % HCA in AOO	204	Larger than the relevant control (AOO)
	205	Larger than the relevant control (AOO)
	206	Larger than the relevant control (AOO)
	207	Larger than the relevant control (AOO)
Vehicle control for the test item: 50 % (v/v) DMSO in water	208	N
	209	N
	210	N
	211	N
Leuco Sulfur Brown 26 presscake 50 % in DMSO	212	N
	213	N
	214	N
	215	N
Leuco Sulfur Brown 26 presscake 25 % in DMSO:water	216	N
	217	N
	218	N
	219	N
Leuco Sulfur Brown 26 presscake 10 % in DMSO:water	220	N
	221	N
	222	N
	223	N
Leuco Sulfur Brown 26 presscake 5 % in DMSO:water	224	N
	225	N
	226	N
	227	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

Dose Group	Measured	Group^*	DPM/Mouse^#	Stimulation
	DPM/group	DPM		Index Values
Vehicle control for the positive control:	3869	3845.0	961.3	1.0
AOO
Positive control:	30513	30489.0	7622.3	7.9
25 % HCA in AOO
Vehicle control for the test item:	1587	1563.0	390.8	1.0
50 % (v/v) DMSO in water
Leuco Sulfur Brown 26 presscake	6929	6905.0	1726.3	4.4
50 % in DMSO
Leuco Sulfur Brown 26 presscake	3927	3903.0	975.8	2.5
25 % in DMSO:water
Leuco Sulfur Brown 26 presscake	3602	3578.0	894.5	2.3
10 % in DMSO:water
Leuco Sulfur Brown 26 presscake	3259	3235.0	808.8	2.1
5 % in DMSO:water

 

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

HCA =a-Hexylcinnamaldehyde

DMSO = Dimethyl sulfoxide

 

*Group DPM = measured DPM_group- average DPM_background

Average DPM_background= 24.0

# 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 item presscake was shown to have no skin sensitisation 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 presscake following dermal exposure in the Local Lymph Node Assay according to OECS guideline 429. The test item was examined in the LLNA as formulations prepared with either Dimethyl sulfoxide (DMSO) or 50 % (v/v) DMSO in water. The maximum achievable concentration resulting in an appropriately applicable homogeneous formulation was 50 % (w/v). The 25 %, 10 % and 5 % (w/v) concentrations were also tested for dose-response analysis.An appropriate positive control (a-Hexylcinnamaldehyde, HCA) and furthermore two negative control groups dosed with 50 % DMSO in water (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 = 7.9), 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. As a sign of a possible irritation significantly increased ear thicknesses (i.e. > 25 % increase compared to the initial values) were observed in the 50 % (w/v) dose group. Ear thicknesses in the other treatment groups did not differ significantly from the initial values or the values measured in the relevant control group during the test. No erythema or other local effects were observed in any treatment group .A significant lymphoproliferative response (SI >= 3) compared to the concurrent control (50 % DMSO) was noted for the test item presscake at a concentration of 50 % (w/v). No significant lymphoproliferative response was observed at the other test concentrations. The observed stimulation index values were 4.4, 2.5, 2.3 and 2.1 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. A statistically significant linear dose-response correlation was observed (p = 0.04, r = 0.96, evaluated by linear regression using the SI values). The increased ear thickness values observed in the 50 % (w/v) dose group may indicate a mild irritation. It is also considered that although 50 % (v/v) DMSO in water was deemed to be the most appropriate negative control for all test item formulation the proportion of DMSO could have been slightly higher in the 50 % (w/v) test item formulation. As DMSO itself usually results in higher proliferation background its higher proportion could result in the overestimation of the SI value in this dose group. According to this the observed SI value of 4.4 is considered as a consequence of an effect other than real sensitization. The stimulation index values of 2.5, 2.3 and 2.1 observed at 25 %, 10 % and 5 % (w/v) concentrations (respectively) did not indicate sensitization potential of the test item. In conclusion, under the conditions of the present assay, the test item presscake tested at the maximum feasible concentration of 50 % (w/v) and also at concentrations of 25 %, 10 % or 5 % (w/v) as adequate homogeneous formulations (suspensions) was shown to have no skin sensitization potential in the Local Lymph Node Assay.