Ethyl 5-sulphamoyl-o-anisate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 12, 2016 - October 25, 2016

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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.51 (Skin sensitisation. local lymph node assay: BrdU-ELISA)

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:

Ethyl 5-sulphamoyl-o-anisate

EC Number:

251-359-7

EC Name:

Ethyl 5-sulphamoyl-o-anisate

Cas Number:

33045-53-3

Molecular formula:

C10H13NO5S

IUPAC Name:

ethyl 2-methoxy-5-sulfamoylbenzoate

Test material form:

solid

Details on test material:

CAS number: 33045-53-3
EC number: 251-359-7
Other name: Ester Sulpiride

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JRj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941Le Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old.
- Weight at study initiation: average weight 0.20 ± 0.01
- Housing: the animals were housed individually (to avoid any test item absorption by oral route) in a suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Tkelad Global 16% Protein Rodent Diet (ENVIGO 2016) ad libitum
- Water: tap water from public distribution system ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas - Eurofins (FRANCE)
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC-25ºC
- Humidity (%): 30%-70%. A relative humidity lower than 30% was registered on 22 October 2016. The minimum value measured was 28%. This deviation is considered as without impact on the conclusion of the study.
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness
- IN-LIFE DATES: From: 12 October 2016 To: 25 October 2016

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

50%, 25% and 10%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test item diluted at 50% in dimethylformamide (DMF) to the dorsal surface of each ear for three consecutive days (Days 1,2,3). The mouse was observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on day 1, day 3 and on day 6. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.
- Compound solubility: A preliminary solubility test was performed using different vehicles (Table 1) and the formulation of 50% in dimethylformamide (DMF) was the most suitable for the test.
- Irritation: No cutaneous reaction was noted at the tested concentration.
- Systemic toxicity: No mortality and no signs of systemic toxicity were noted.
- Ear thickness measurements: values were within the acceptable range (Table 2)
- Erythema scores: no signs of erithema were observed (Table 2)

MAIN STUDY
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in dimethylformamide based on the results of the pre-screen tests. The mice treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Clinical observations. all animals were observed daily on Days 1, 2, 3 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: the bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighted in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighted.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensitisation. Local Lymph Node Assay:BrdU-ELISA
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 - Batch No. 11866200). Briefly, 100 µL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as: BrdU labelling index = (ABSem - ABS blankem) - (ABSref - ABS blankref)
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive. Any test item failing to produce a SI>1.6 will be classified as a "sensitiser".
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was detemined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6 -fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 - d) / (b - d)] x (a - c)
Legend: a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared in dimethylformamide. The test item was diluted at 50% (maximum concentration available), 25% and 10% in dimethylformamine. The mice were treated by daily application of 25 µL of the appropiate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1,2,3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
On day 5 (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
The BrdU solution was prepared by weighing 309.5 mg of 5 -bromo-2'-deoxyuridine 8SIGMA - Batch No. HMBD6482V) in a glass sample bottle and adding 30.98 mL of physiological saline. Then, the preparation was stirred magnetically and vortex, just before treatment.
On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and adminitration continued to fatal levels. The draining auricular lymph nodes from the four mic were excised.
From each mouse, a single-cell suspension through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of PBD (Ca2 +/Mg2+ - free) into a well of multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 -0.2. Then, BrdU was determined.

Positive control substance(s):

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

Results and discussion

Positive control results:

EC1.6= 9.48%. The substance has to be classified in category 1 "Skin sensitisation".

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.04

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

ca. 0.91

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

ca. 1.37

Test group / Remarks:

50%

Results 4

Parameter:

other: EC1.6

Remarks on result:

not determinable

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50% (Tables 7 and 8).

DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index of more than 1.6 was recorded whatever the tested concentration. the Stimulation Index (SI) calculated by individual approach was 1.04, 0.91 and 1.37 for the treated groups at 10%, 25% and 50%, respectively (Tables 5 and 6).

EC3 CALCULATION
The EC1.6 cannot be determined in this study.

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test (Table 3).
Slight dryness of the skin was noted in all animals at the tested concentrations of 25% on day 3. No other cutaneous reaction was noted whatever the tested concentration.
Therefore, the test item has to be considered as not excessively irritant at these concentrations.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period (Table 2).

Any other information on results incl. tables

Table 1. Vehicle determination record

 

Vehicle	Concentration	Method of preparation	Description of formulation	Suitability*
AOO	50%	0.1 g test item + 0.1 g vehicle	Non homogenous suspension	No
DMF	75%	1.5 g test item + 0.5 g vehicle	Non homogenous whitish paste	No
DMF	50%	0.1 g test item + 0.1 g vehicle	Colorless solution	Yes
DMF	10%	0.2 g test item + 0.18 g vehicle	Colorless solution	Yes
MEK	50%	0.2 g test item + 0.2 g vehicle	Non homogenous suspension	No
PG	50%	0.2 g test item + 0.2 g vehicle	Non homogenous suspension	No
DMO	50%	0.5 g test item + 0.5 g vehicle	Non homogenous suspension	No
Pluronic®1% acid	50%	0.2 g test item + 0.2 g vehicle	Non homogenous suspension	No

 

Table 2. Clinical observation, bodyweight and mortality data

 

Concentration %	Animal	Bodyweight (g)		DAY
		Day 1	Day 6	1	2	3	4	5	6
50%	Sf8719	19.0	19.4	0	0	0^o	0	0	0

0: No sign of systemic toxicity and no sign of erythema

^o: residue of the test item

 

	Ear thickness (mm) on day 1	Ear thickness (mm) on day 3	Ear thickness (mm) on day 6	Ear weight (mg) on day 6	Weight Lymph nodes (mg)
Sf8719	0.19	0.20	0.20	25.4	5.2

 

Main test:

Table 3. Individual clinical observation and mortality data

 

Groups	Test item	Amimals	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	DMF	Nº Sf 8743	0	0	0	0	0	0
		Nº Sf 8744	0	0	0	0	0	0
		Nº Sf 8745	0	0	0	0	0	0
		Nº Sf 8746	0	0	0	0	0	0
2	10%	Nº Sf 8748	0	0	0	0	0	0
		Nº Sf 8752	0	0	0	0	0	0
		Nº Sf 8750	0	0	0	0	0	0
		Nº Sf 8751	0	0	0	0	0	0
3	25%	Nº Sf 8753	0	0	0^o*	0^o	0^o	0^o
		Nº Sf 8754	0	0	0^o*	0^o	0^o	0^o
		Nº Sf 8757	0	0	0^o*	0^o	0^o	0^o
		Nº Sf 8756	0	0	0^o*	0^o	0^o	0^o
4	50%	Nº Sf 8758	0	0^o	0^o	0^o	0^o	0^o
		Nº Sf 8759	0	0^o	0^o	0^o	0^o	0^o
		Nº Sf 8760	0	0^o	0^o	0^o	0^o	0^o
		Nº Sf 8761	0	0^o	0^o	0^o	0^o	0^o

0: No sign of systemic toxicity and no sign of erythema

DMF. dimethylformamide

^o: residue of the test item

*: slight dryness

 

Table 4. Individual body weight and body weight gain

 

Groups	Test item	Amimals	Body weight (g)		Body weight gain (g)
			Day 1	Day 6
1	DMF	Nº Sf 8743	20.8	22.2	1.4
		Nº Sf 8744	21.3	23.7	2.4
		Nº Sf 8745	20.6	21.4	0.8
		Nº Sf 8746	21.3	23.1	1.8
	MEAN		21.0	22.6	1.6
	Standard-deviation		0.4	1.0	0.7
2	10%	Nº Sf 8748	21.0	21.8	0.8
		Nº Sf 8752	18.4	20.6	2.2
		Nº Sf 8750	21.6	22.3	0.7
		Nº Sf 8751	22.1	22.9	0.8
	MEAN		20.8	21.9	1.1
	Standard-deviation		1.6	1.0	0.7
3	25%	Nº Sf 8753	21.1	21.0	-0.1
		Nº Sf 8754	19.2	20.7	1.5
		Nº Sf 8757	18.4	19.0	0.6
		Nº Sf 8756	20.8	22.1	1.3
	MEAN		19.9	20.7	0.8
	Standard-deviation		1.3	1.3	0.7
4	50%	Nº Sf 8758	20.6	22.7	2.1
		Nº Sf 8759	20.9	21.3	0.4
		Nº Sf 8760	19.1	19.2	0.1
		Nº Sf 8761	19.3	19.9	0.6
	MEAN		20.0	20.8	0.8
	Standard-deviation		0.9	1.6	0.9

DMF. dimethylformamide

Table 5. BrdU index & Stimulation index per group and calculation of EC_1.6

 

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	DMF	0.655	n.a.	n.a.	n.a.
2	10%	0.681	1.04±0.28	negative	n.a.
3	25%	0.596	0.91±0.37	negative
4	50%	0.894	1.37±0.09	negative

 

Table 6.BrdU index & Stimulation index (individual data)

Groups	Test item	Amimal	BrdU-Index (DO Indiv)	BrdU-Index (DO mean)	BrdU-Index mean*	Stimulation Index S.I. (indiv±Standard deviation
1	DMF	Sf 8743	0.988	0.923	0.655	n.a
			0.937
			0.844
		Sf 8744	0.626	0.602		n.a
			0.569
			0.611
		Sf 8745	0.537	0.538		n.a
			0.506
			0.571
		Sf 8746	0.604	0.555		n.a
			0.613
			0.446
2	10%	Sf 8748	0.891	0.880	0.681	1.34±0.09
			0.933
			0.816
		Sf 8752	0.823	0.797		1.22±0.04
			0.778
			0.790
		Sf 8750	0.518	0.515		0.79±0.06
			0.474
			0.553
		Sf 8751	0.594	0.531		0.81±0.09
			0.521
			0.477
3	25%	Sf 8753	0.945	0.956	0.596	1.46±0.02
			0.953
			0.968
		Sf 8754	0.515	0.509		0.78±0.03
			0.484
			0.526
		Sf 8757	0.469	0.489		0.75±0.05
			0.472
			0.525
		Sf 8756	0.570	0.431		0.66±0.19
			0.323
			0.399
4	50%	Sf 8758	0.930	0.926	0.894	1.41±0.02
			0.934
			0.913
		Sf 8759	0.785	0.811		1.24±0.03
			0.820
			0.826
		Sf 8760	0.841	0.905		1.38±0.09
			0.911
			0.963
		Sf 8761	0.931	0.932		1.42±0.16
			1.033
			0.830

*: mean:Σindividual value / 4

DMF: dimethylformamide

Table 7. Individual Ear thickness and irritation level.

 

Groups	Test item	Amimals	Day 1 Ear thickness (mm)	Day 3 Ear thickness (mm)	Day 6 Ear thickness (mm)	Ear thickness increase D3/D1 (%)	Ear thickness increase D6/D1 (%)
1	DMF	Nº Sf 8743	0.19	0.21	0.21	10.5	10.5
		Nº Sf 8744	0.21	0.21	0.21	0.0	0.0
		Nº Sf 8745	0.19	0.19	0.20	0.0	5.3
		Nº Sf 8746	0.19	0.19	0.21	0.0	10.5
	MEAN		0.20	0.20	0.21	2.6	6.6
	Standard-deviation		0.01	0.01	0.00	5.3	5.0
2	10%	Nº Sf 8748	0.19	0.19	0.19	0.0	0.0
		Nº Sf 8752	0.19	0.19	0.21	0.0	10.5
		Nº Sf 8750	0.20	0.20	0.21	0.0	5.0
		Nº Sf 8751	0.19	0.19	0.20	0.0	5.3
	MEAN		0.19	0.19	0.20	0.0	5.2
	Standard-deviation		0.01	0.01	0.01	0.0	4.3
3	25%	Nº Sf 8753	0.20	0.20	0.21	0.0	5.0
		Nº Sf 8754	0.19	0.19	0.20	0.0	5.3
		Nº Sf 8757	0.19	0.20	0.20	5.3	5.3
		Nº Sf 8756	0.19	0.19	0.19	0.0	0.0
	MEAN		0.19	0.20	0.20	1.3	3.9
	Standard-deviation		0.01	0.01	0.01	2.6	2.6
4	50%	Nº Sf 8758	0.20	0.21	0.21	5.0	5.0
		Nº Sf 8759	0.19	0.21	0.21	10.5	10.5
		Nº Sf 8760	0.19	0.21	0.21	10.5	10.5
		Nº Sf 8761	0.19	0.19	0.20	0.0	5.3
	MEAN		0.19	0.21	0.21	6.5	7.8
	Standard-deviation		0.01	0.01	0.00	5.1	3.1

DMF: dimethylformamide

Table 8. Individual Ear biopsy weight and lymph node weight.

 

Groups	Test item	Amimals	Ear weight Day 6 (mg)	% of ear weight increased/group1	Lymph nodes (mg)
1	DMF	Nº Sf 8743	25.3		6.7
		Nº Sf 8744	24.5		4.4
		Nº Sf 8745	23.9		5.2
		Nº Sf 8746	25.9		4.1
	MEAN		24.9		5.1
	Standard-deviation		0.9		1.2
2	10%	Nº Sf 8748	23.5	-2.8	4.9
		Nº Sf 8752	23.4		4.8
		Nº Sf 8750	25.8		5.8
		Nº Sf 8751	24.1		5.1
	MEAN		24.2		5.2
	Standard-deviation		1.1		0.5
3	25%	Nº Sf 8753	23.4	-3.3	4.6
		Nº Sf 8754	24.8		5.5
		Nº Sf 8757	24.1		6.3
		Nº Sf 8756	24.0		6.0
	MEAN		24.1		5.6
	Standard-deviation		0.6		0.7
4	50%	Nº Sf 8758	23.5	-0.6	6.2
		Nº Sf 8759	26.3		8.2
		Nº Sf 8760	23.8		6.3
		Nº Sf 8761	25.4		6.7
	MEAN		24.8		6.9
	Standard-deviation		1.3		0.9

DMF: dimethylformamide

Table 9. Summary of result – skin irritation

 

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation#
1	DMF	6.6	n.a	No
2	10%	5.2	-2.8	No
3	25%	3.9	-3.3	No
4	50%	7.8	-0.6	No

#: O.E.C.D. criteria: (% increase in ear thickness lower than 25%, score of erythema lower than 3)

DMF: dimethylformamide

Table 10. BrdU index & Stimulation index per group and calculation of EC_1.6of the positive control (study performed 29 June 2016 – 5 July 2016)

 

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	AHO	1.028	n.a.	n.a.	n.a.
2	5%	1.379	1.34±0.10	positive	9.48%
3	10%	1.680	1.63±0.19	positive
4	25%	2.009	1.95±0.41	positive

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show skin sensitisation potential unter the tested conditions in the LLNA assay (OECD 442B). The stimulation indexes were 1.04, 0.91 and 1.37 at concentrations of test item 10%, 25% and 50%, respectively.

Executive summary:

The skin sensitisation potential of the test item was tested according OECD 442B and E.U. B.51 method, following GLP. It was tested on three groups of four mouse CBA/J treated for three consecutive days with 50 µL (25 µL per ear) of the test item diluted at concentrations of 10%, 25% and 50% in dimethylformamide. A control group was treated with dimethylformamide. On day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected by the intraperitoneal route. On day 6, the profileration of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item has to be considered as not excessively irritant at these concentrations. The Stimulation Index (SI) calculated by individual approach was 1.04, 0.91 and 1.37 for the treated groups at 10%, 25% and 50%, respectively. The EC1.6 cannot be determined in this study.