N,N'-(ethoxymethylsilylene)bis[N-methylbenzamide]

Endpoint summary

Description of key information

In vivo skin sensitisation

Key study: Study according OECD 442B and EU B.51. GLP study. The Stimulation Index (SI) calculated by individual approach were 1.33, 0.78 and 1.66 at concentrations of test item 10%, 25% and 50%, respectively. The EC1.6 determined was 19%. Therefore, has to be classified as a sensitiser in Category 1 (sub-category 1B) according to CLP Regulation EC 1272/2008.

In vitro skin sensitization

Data waiving: Study scientifically not necessary / other information avilable. An in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2, 2016 - December 6, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.

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 or 9 weeks old.
- Weight at study initiation: average weight 20.8 ± 1.1 grams
- 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: Teklad 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. A temperature lower than 19ºC was registered on 02 & 03 December 2016. The minimum value measured was 18ºC. This deviation is considered as without impact on the conclusion of the study.
- Humidity (%): 30%-70%. A relative humidity lower than 30% was registered on 02 & 03 December 2016. The minimum value measured was 26%. 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: 2 November 2016 To: 6 December 2016

Vehicle:

acetone/olive oil (4:1 v/v)

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 (100%) 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.
Due to the severe skin reaction noted at the test dose 100%, two other mice were treated by daily application of 25 µL of the test item diluted at 25% and 10% in Acetone/olive oil (4:1, v/v), respectively, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed daily form 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 mice was recorded on Day 1 (prior to dosing) and Day 6.
Due to the absence of skin irritation at the test doses 10% and 25%, another mouse was treated by daily application of 25 µL of the test item diluted at 50% in Acetone/olive oil (4:1, v/v), respectively, 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 (Table 1) and the formulation of 50% in Acetone/olive oil (AOO) was the most suitable for the test. Although residual test item was noted from day 2 to day 6 at the tested concentration of 50%, it was chosen as the highest concentration for the mail study.
- Irritation: No cutaneous reaction was noted at the tested concentrations of 50%, 25% and 10%. Sign of excessive irritation was noted at the tested concentration of 100%.
- 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). Dryness of the skin was noted on day 2 and scab from day 3 to day 6, at the tested concentration of 100%.

MAIN STUDY
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in Acetone/olive oil (4:1, v/v) based on the results of the pre-screen tests. The mice were 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. 17267000). 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 added to each well and allowed to react. Subsequently the 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 "non-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

Interpretation of the results according to CLP regulation (EC No. 1272/2008):
If at least one concentration of the test item results in a SI ≥3, it should be classified as "sensitiser".
If the EC value ≤ 2, the test item will be classified in "sub-category 1A".
If the EC value > 2, the test item will be classified in "sub-category 1B".

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared in Acetone/olive oil (4:1, v/v). Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in Acetone/olive oil (4:1, v/v). 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 400.6 mg of 5 -bromo-2'-deoxyuridine (SIGMA - Batch No. HMBD6482V) in a glass sample bottle and adding 40.06 mL of physiological saline. Then, the preparation was magnetically stirred, 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 mice were excised.
From each mouse, a single-cell suspension through of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of PBS (Ca2 +/Mg2+ - free) into a well of a 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)

Positive control results:

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

Key result

Parameter:

SI

Value:

ca. 1.33

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

ca. 1.78

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

ca. 1.66

Test group / Remarks:

50%

Parameter:

other: EC1.6

Remarks:

(%)

Value:

19

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).
The result obtained may be considered as a borderline result (i.e. positive response between and SI of 1.6 and 1.9); furthermore, no dose response was noted.

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).

Table 1. Vehicle determination record

 

Vehicle	Concentration	Method of preparation	Description of formulation	Suitability*
AOO	50%	0.3 mL test item + 0.3 mL vehicle	Yellow solution	Yes
AOO	10%	0.2 mL test item at 50% + 0.8 mL vehicle	Yellow solution	Yes

*: suitable for dosing if formulation is a solution or fine homogenous suspension which can be administered via a micropipette

 

AOO: Acetone/olive oil (4:1; v/v)

 

Table 2. Clinical observation, bodyweight and mortality data

 

Concentration %	Animal	Bodyweight (g)		DAY
		Day 1	Day 6	1	2	3	4	5	6
100%	Sf8867	21.7	22.8	0	0**	0***	0***	0***	0***
50%	Sf8990	22.1	22.7	0	0^o	0^o	0^o	0^o	0^o
25%	Sf8918	19.2	20.7	0	0	0	0	0	0
10%	Sf8917	23.7	24.9	0	0	0	0	0	0

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

**: dryness, ***: scab

^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 thickness increase D3/D1 (%)	Ear weight (mg) on day 6	Weight Lymph nodes (mg)
Sf8867	0.21	0.73	0.75	+257	68.4	10.1
Sf8990	0.22	0.22	0.22	0	25.1	9.8
Sf8918	0.20	0.21	0.21	+5	28.6	9.9
Sf8917	0.20	0.22	0.21	+5	27.9	6.9

 

The concentration of 50% was chosen as the highest concentration for the main study.

 

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	AOO	Nº Sf 9024	0	0	0	0	0	0
		Nº Sf 9025	0	0	0	0	0	0
		Nº Sf 9026	0	0	0	0	0	0
		Nº Sf 9027	0	0	0	0	0	0
2	10%	Nº Sf 9029	0	0	0	0	0	0
		Nº Sf 9030	0	0	0	0	0	0
		Nº Sf 9031	0	0	0	0	0	0
		Nº Sf 9032	0	0	0	0	0	0
3	25%	Nº Sf 9034	0	0	0	0	0	0
		Nº Sf 9035	0	0	0	0	0	0
		Nº Sf 9036	0	0	0	0	0	0
		Nº Sf 9037	0	0	0	0	0	0
4	50%	Nº Sf 9039	0	0	0^o	0	0	0
		Nº Sf 9040	0	0	0^o	0	0	0
		Nº Sf 9041	0	0	0^o	0	0	0
		Nº Sf 9042	0	0	0^o	0	0	0

0: No sign of systemic toxicity

AOO: Acetone/olive oil

^o: residue of the test item

 

 

Table 4. Individual body weight and body weight gain

 

Groups	Test item	Amimals No.	Bodyweight (g)		Bodyweight gain (g)
			Day 1	Day 6
1	AOO	Nº Sf 9024	22.1	23.7	1.6
		Nº Sf 9025	20.0	20.8	0.8
		Nº Sf 9026	20.3	21.2	0.9
		Nº Sf 9027	20.4	21.1	0.7
	MEAN		20.7	21.7	1.0
	Standard-deviation		0.9	1.3	0.4
2	10%	Nº Sf 9029	19.8	20.5	0.7
		Nº Sf 9030	19.7	20.5	0.8
		Nº Sf 9031	21.5	22.3	0.8
		Nº Sf 9032	20.3	20.7	0.4
	MEAN		20.3	21.0	0.7
	Standard-deviation		0.8	0.9	0.2
3	25%	Nº Sf 9034	20.4	20.8	0.4
		Nº Sf 9035	23.4	23.1	-0.3
		Nº Sf 9036	22.0	22.8	0.8
		Nº Sf 9037	21.1	21.1	0.0
	MEAN		21.7	22.0	0.2
	Standard-deviation		1.3	1.2	0.5
4	50%	Nº Sf 9039	19.6	21.2	1.6
		Nº Sf 9040	22.2	23.5	1.3
		Nº Sf 9041	20.9	21.6	0.7
		Nº Sf 9042	19.6	19.5	-0.1
	MEAN		20.6	21.5	0.9
	Standard-deviation		1.2	1.6	0.8

AOO: Acetone/olive oil

 

 

 

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	AOO	0.826	n.a.	n.a.	n.a.
2	10%	1.100	1.33±0.31	negative	19.00%
3	25%	1.472	1.78±0.11	positive
4	50%	1.367	1.66±0.04	positive

 

 

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	AOO	Sf 9024	0.747	0.749	0.826	n.a
			0.731
			0.769
		Sf 9025	0.926	0.915		n.a
			0.890
			0.929
		Sf 9026	0.928	0.933		n.a
			0.920
			0.951
		Sf 9027	0.766	0.706		n.a
			0.667
			0.684
2	10%	Sf 9029	1.376	1.288	1.100	1.56±0.13
			1.171
			1.318
		Sf 9030	1.384	1.348		1.63±0.05
			1.302
			1.358
		Sf 9031	0.957	0.889		1.08±0.14
			0.956
			0.755
		Sf 9032	0.812	0.876		1.06±0.07
			0.884
			0.931
3	25%	Sf 9034	1.494	1.496	1.472	1.81±0.03
			1.524
			1.471
		Sf 9035	1.778	1.554		1.88±0.24
			1.428
			1.456
		Sf 9036	1.618	1.490		1.80±0.14
			1.394
			1.458
		Sf 9037	1.313	1.346		1.63±0.08
			1.308
			1.418
4	50%	Sf 9039	1.412	1.413	1.367	1.71±0.08
			1.476
			1.350
		Sf 9040	1.406	1.358		1.64±0.19
			1.483
			1.184
		Sf 9041	1.311	1.354		1.64±0.05
			1.380
			1.371
		Sf 9042	1.281	1.344		1.63±0.08
			1.337
			1.414

*: mean:Σindividual value / 4

AOO: Acetone/olive oil

 

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	AOO	Nº Sf 9024	0.21	0.20	0.22	-4.8	4.8
		Nº Sf 9025	0.20	0.21	0.20	5.0	0.0
		Nº Sf 9026	0.20	0.20	0.20	0.0	0.0
		Nº Sf 9027	0.22	0.20	0.20	-9.1	-9.1
	MEAN		0.21	0.20	0.21	-2.2	-1.1
	Standard-deviation		0.01	0.00	0.01	6.1	5.8
2	10%	Nº Sf 9029	0.20	0.21	0.19	5.0	-5.0
		Nº Sf 9030	0.20	0.21	0.20	5.0	0.0
		Nº Sf 9031	0.21	0.22	0.21	4.8	0.0
		Nº Sf 9032	0.20	0.20	0.19	0.0	-5.0
	MEAN		0.20	0.21	0.20	3.7	-2.5
	Standard-deviation		0.00	0.01	0.01	2.5	2.9
3	25%	Nº Sf 9034	0.20	0.20	0.21	0.0	5.0
		Nº Sf 9035	0.21	0.21	0.20	0.0	-4.8
		Nº Sf 9036	0.19	0.20	0.20	5.3	5.3
		Nº Sf 9037	0.21	0.21	0.21	0.0	0.0
	MEAN		0.20	0.21	0.21	1.3	1.4
	Standard-deviation		0.01	0.01	0.01	2.6	4.8
4	50%	Nº Sf 9039	0.21	0.22	0.22	4.8	4.8
		Nº Sf 9040	0.19	0.22	0.22	15.8	15.8
		Nº Sf 9041	0.21	0.22	0.22	4.8	4.8
		Nº Sf 9042	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.22	0.22	6.3	6.3
	Standard-deviation		0.01	0.01	0.01	6.7	6.7

AOO: Acetone/olive oil

 

  

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	AOO	Nº Sf 9024	27.5		5.8
		Nº Sf 9025	28.9		5.2
		Nº Sf 9026	28.8		5.0
		Nº Sf 9027	28.6		5.2
	MEAN		28.5		5.3
	Standard-deviation		0.6		0.3
2	10%	Nº Sf 9029	28.7	-3.4	6.8
		Nº Sf 9030	28.4		6.9
		Nº Sf 9031	26.0		7.3
		Nº Sf 9032	26.8		8.5
	MEAN		27.5		7.4
	Standard-deviation		1.3		0.8
3	25%	Nº Sf 9034	27.5	-5.1	8.7
		Nº Sf 9035	27.0		10.1
		Nº Sf 9036	27.4		8.8
		Nº Sf 9037	26.1		9.0
	MEAN		27.0		9.2
	Standard-deviation		0.6		0.6
4	50%	Nº Sf 9039	30.3	4.4	9.2
		Nº Sf 9040	29.4		9.1
		Nº Sf 9041	28.8		8.4
		Nº Sf 9042	30.3		8.4
	MEAN		29.7		8.8
	Standard-deviation		0.7		0.4

AOO: Acetone/olive oil

 

 

Table 9. Summary of result – skin irritation

 

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation#
1	AOO	-1.1	n.a	No
2	10%	-2.5	-3.4	No
3	25%	1.4	-5.1	No
4	50%	6.3	4.4	No

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

AOO: Acetone/olive oil

 

 

 

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

AHO:α-Hexylcinnamaldehyde

 

 

 

 

 

 

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item has to be classified as a skin sensitiser in category 1 (sub-category 1B), under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 1.33, 0.78 and 1.66 at concentrations of test item 10%, 25% and 50%, respectively. The EC1.6 determined was 19%.

Executive summary:

The skin sensitisation potential of the test item was tested according OECD 442B and E.U. B.51 method, following GLP. Firstly, a preliminary study was conducted, the test item was applied undiluted (100%) for three days. Severe skin reaction was noted so the test item was applied diluted in the main test. 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 Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was deremined 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. Residual test item was noted in all animals treated at 50% on day 3. No increase in ear thickness and in ear weight was noted in animals treated at 50%, 25% and 10%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 1.33, 1.78 and 1.66 for the treated groups at 10%, 25% and 50% respectively. The EC1.6 determined by linear regression was 19% (which is > 2%). The result obtained may be considered as a borderline result (i.e. positive response between and SI of 1.6 and 1.9); furthermore, no dose response was noted. Under these experimental conditions, the test item has to be classified as a sensitiser in Category 1 (sub-category 1B), in accordance with the CLP Regulation (EC) no. 1272/2008.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Study scientifically not necessary / other information available. An in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo skin sensitisation

Key study: 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 Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was deremined 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. Residual test item was noted in all animals treated at 50% on day 3. No increase in ear thickness and in ear weight was noted in animals treated at 50%, 25% and 10%. The Stimulation Index (SI) calculated by individual approach was 1.33, 1.78 and 1.66 for the treated groups at 10%, 25% and 50% respectively. The EC1.6 determined by linear regression was 19%. Under these experimental conditions, the test item has to be classified as a sensitiser in Category 1 (sub-category 1B), in accordance with the CLP Regulation(EC) no. 1272/2008.

In vitro skin sensitisation

Data waiving: Study scientifically not necessary / other information avilable. Data from a LLNA study according to OECD 422B was available. Therefore, an in vitro study was not deemed necessary.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information (EC1.6 = 19 % in OECD 442B test), the test item has to be classified as a skin sensitiser Category 1 (sub-category 1B), H317: May cause an allergic skin reaction, according to CLP Regulation 1272/2008.