N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Part of weight-of-evidence approach adapting the information requirements of Annex VII 8.3.1 and 8.3.2. under REACH in accordance with Annex XI Section 1.2. A sequential series of skin sensitisation tests were performed which collectively provide all the information required to satisfy the information endpoint for Annex VII 8.3.1. and 8.3.2 under REACH. Therefore in accordance with Annex XI, 1.2 of the REACH Regulation is no additional testing is scientifically necessary based on a weight-of evidence approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Version / remarks:

Appendix IA, June 25 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

EC Number:

876-151-9

Cas Number:

2292123-68-1

Molecular formula:

C27H24N2O7S2

IUPAC Name:

N,N’-di[3-(p-toluene sulfonyl)oxy]phenyl urea

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks for pre-screen, 9 weeks for main study
- Weight at study initiation: See Main study Day 1 - Table 3 in results sections
- Housing: polycarbonate cages with wood chips and environmental enrichment
- Diet (e.g. ad libitum): pelleted diet, ad libitum
- Water (e.g. ad libitum): chlorinated water, ad libitum
- Acclimation period: 7 days for pre-screen, 14 days for main study
- Indication of any skin lesions: all animals in good health condition

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

10%, 25% and 50%

No. of animals per dose:

4 animals per test item concentration
4 animals for vehicle control
4 animals for positive control

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Vehicle solubility trials were performed. Recommended vehicles in the OECD 442, acetone: olive oil (4:1 v/v/ AOO), DMF, methyl ethyl ketone (MEK) and dimethylsulfoxide (DMSO) were selected for solubility trials. The test item dissolved in DMF and DMSO at 50% (v/v), however, after 5 hours test item crystallised in the DMSO. The test item was not soluble on MEK or AOO. and therefore DMF selected as the vehicle for the pre-screen and main study.
- Irritation: none
- Systemic toxicity: none (no abnormalities reported)
- Ear thickness measurements: yes (see Table 2 in the results section)
- Erythema scores: no erythema so not scored

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item was regarded as a "sensitiser" when the SI of the test item was 2.0 or more and was regarded as "non-sensitiser" when the SI of the test item group was less than 1.6. When the SI was between 1.6 and 1.9, dose-response relationship and statistical significance would be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
The test solution was prepared on each sensitisation day. 0.5 was dissolved in DMF and filled up to make 1mL of 50 w/v% solution. The 50 w/v% solution was serially diluted to prepare the test solutions at 25 and 10 w/v%.
25µL of each formulation was applied to the dorsum of each ear of the animals using a micropipette once per day for 3 consecutive days.
Approx. 48 hours after the final application of the formulations, 0.5 mL of BrdU solution was administrated intraperitoneally to each animal using a syringe and a needle. Approx. 24 hours after the BrdU administration, animals were humanely killed and each auricular lymph node was taken. The lymph nodes were carefully dissected and trimmed of surrounding tissue and fat, weighed both sides together. The mean values and standard deviations of the local lymph node weights were calculated for each group. The lymph nodes were stored individually in a biomedical freezer.

Positive control substance(s):

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

Statistics:

No statistic performed as SI values all <1.6.

Results and discussion

Positive control results:

SI = 2.93 +/-0.24.
The SI value was >2 and therefore indicates that the test system was functioning as intended.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.08

Variability:

S.E. +/- 0.12

Test group / Remarks:

10%

Remarks on result:

other: Mean SI value

Results 2

Parameter:

SI

Value:

1.18

Variability:

S.E. +/- 0.15

Test group / Remarks:

25%

Remarks on result:

other: Mean SI value

Results 3

Parameter:

SI

Value:

1.48

Variability:

S.E. +/- 0.21

Test group / Remarks:

50%

Remarks on result:

other: Mean SI value

Results 4

Parameter:

SI

Value:

2.93

Variability:

S.E. +/- 0.24

Test group / Remarks:

Positive control

Remarks on result:

other: Mean SI value

Results 5

Parameter:

SI

Value:

1

Variability:

S.E. +/- 0.04

Test group / Remarks:

Vehicle control

Remarks on result:

other: Mean SI value

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Lymph node weight - see Table 5

DETAILS ON STIMULATION INDEX CALCULATION


EC3 CALCULATION: not calculated

CLINICAL OBSERVATIONS:
See Table 4

BODY WEIGHTS
See Table 3

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness). None.

Any other information on results incl. tables

Table 1: Body weights in the pre-screen test

Exp. group		Animal No.	Body weights (g)
Group	Concentration (w/v%)		Day 1	Day 6^a)
Test item	10.0	1	22.1	22.2 (100.5)
	25.0	2	22.4	23.3 (104.0)
	50.0	3	22.4	23.1 (103.1)

Figures in parentheses indicate percentages compared to the initial body weight (Day 1)

 

Table 2: Thicknesses of auricle in the pre-screen test

Exp. Group		Animal No.	Thickness of auricle (mm)
Group	Concentration (w/v%)		Day 1		Day 3^a)		Day 6^a)
Test item			Left	Right	Left	Right	Left	Right
	10.0	1	0.185	0.190	0.205 (110.8)	0.210 (110.8)	0.190 (102.7)	0.205 (107.9)
	25.0	2	0.205	0.195	0.195 (95.1)	0.210 (107.7)	0.210 (102.4)	0.210 (107.7)
	50.0	3	0.180	0.190	0.200 (111.1)	0.210 (110.5)	0.200 (111.1)	0.195 (102.6)

Figures in parentheses indicate percentages compared to the initial thicknesses (Day 1)

 

Table 3: Body weights in the main study

Exp Group		Animal No.	Body weights (g)
Group	Concentration (w/v%)		Day 1		Day 6
			Individual	Mean ± S.D.	Individual	Mean ± S.D.
Vehicle Control (DMF)	-	1	22.3	22.43 ±1.80	22.8	23.15 ± 1.88
		2	25.0		25.9
		3	21.4		21.8
		4	21.0		22.1
Positive Control (HCA)	25.0	5	24.7	23.45 ± 2.43	24.4	23.35 ± 1.92
		6	26.2		25.4
		7	20.9		21.1
		8	22.0		22.5
Test item	10.0	9	24.5	22.90 ± 1.49	23.9	22.50 ± 1.27
		10	23.8		23.0
		11	21.9		20.9
		12	21.4		22.2
	25.0	13	22.2	22.55 ± 1.84	23.0	22.70 ± 1.22
		14	25.2		24.2
		15	21.0		21.3
		16	21.8		22.3
	50.0	17	22.5	22.45 ± 1.40	23.7	22.80 ± 1.34
		18	24.0		24.1
		19	20.6		21.2
		20	22.7		22.2

S.D: Standard deviation

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

 

Table 4: Clinical signs in the main study

Exp Group		Animal No.	Observation period
Group	Concentration (w/v%)
			Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
Vehicle Control (DMF)	-	1	-	-	-	-	-	-
		2	-	-	-	-	-	-
		3	-	-	-	-	-	-
		4	-	-	-	-	-	-
Positive Control (HCA)	25.0	5	-	-	-	-	-	-
		6	-	-	-	-	-	-
		7	-	-	-	-	-	-
		8	-	-	-	-	-	-
Test item	10.0	9	-	-	-	-	-	-
		10	-	-	-	-	-	-
		11	-	-	-	-	-	-
		12	-	-	-	-	-	-
	25.0	13	-*	-*	-*	-	-	-
		14	-*	-*	-*	-	-	-
		15	-*	-*	-*	-	-	-
		16	-*	-*	-*	-	-	-
	50.0	17	-*	-*	-*	-	-	-
		18	-*	-*	-*	-	-	-
		19	-*	-*	-*	-	-	-
		20	-*	-*	-*	-	-	-

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

- : no abnormalities detected

*-: test item crystallised on ear (after application)

 

Table 5: Lymph node weights in the main study

Exp Group		Animal No.	Lymph node weights (g)
Group	Concentration (w/v%)
			Individual	Mean ± S.D.
Vehicle Control (DMF)	-	1	4.3	4.28 ± 0.21
		2	4.5
		3	4.0
		4	4.3
Positive Control (HCA)	25.0	5	9.1	8.33 ± 0.80
		6	8.5
		7	7.2
		8	8.5
Test item	10.0	9	4.7	4.65 ± 0.34
		10	4.5
		11	4.3
		12	5.1
	25.0	13	4.7	4.35 ± 0.39
		14	4.4
		15	4.5
		16	3.8
	50.0	17	4.7	5.20 ± 0.36
		18	5.4
		19	5.2
		20	5.5

S.D: Standard deviation

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

 

Table 6: BrdU labelling indices and stimulation indices in the main study

Exp Group		Animal No.	BrdU labelling index		Stimulation index
Group	Concentration (w/v%)
			Individual	Mean ± S.E.	Individual	Mean ± S.E.
Vehicle Control (DMF)	-	1	0.176	0.1560 ± 1.80	1.1	1.00 ± 0.04
		2	0.153		1.0
		3	0.136		0.9
		4	0.159		1.0
Positive Control (HCA)	25.0	5	0.568	0.4570 ± 0.0395	3.6	2.93 ± 0.24
		6	0.384		2.5
		7	0.426		2.7
		8	0.450		2.9
Test item	10.0	9	0.216	0.1708 ± 0.0169	1.4	1.08 ± 0.12
		10	0.143		0.9
		11	0.177		1.1
		12	0.147		0.9
	25.0	13	0.233	0.1835 ± 0.0215	1.5	1.18 ± 0.15
		14	0.185		1.2
		15	0.188		1.2
		16	0.128		0.8
	50.0	17	0.169	0.2285 ± 0.0322	1.1	1.48 ± 0.21
		18	0.243		1.6
		19	0.189		1.2
		20	0.313		2.0

S.E: Standard error

DMF: N,N-dimethylformamide

HCA: α-hexylcinnamaldehyde

Applicant's summary and conclusion

Conclusions:

The SIs of the 50.0, 25.0 and 10.0 w/v% test item groups were 1.48, 1.18 and 1.08 (all SIs less than the cut-off value of 1.6. Therefore under the condition of the test, the test item was judged to be non-sensitiser.