Ethyl ethyl(phenyl)carbamate

Endpoint summary

Description of key information

Skin sensitisation in vivo

Key study: OECD TG 442 -B and GLP study. The stimulation index of the test item is 0.81, 1.01 and 1.08 at the concentration of 25%, 50% and 100% respectively, on a LLNA study in mouse. As the value of stimulation index is lower than 1.6, the test item is not a sensitizer.

Data waiving: In vitro study is not necessary because the study in vivo (LLNA) is already 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 - November 22, 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: test method B.51

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
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle:yes, in Acetone/olive oil (4:1, v/v).

Species:

mouse

Strain:

CBA:J

Remarks:

(CBA/JRj) strain mice (SPF caw)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:Elevage Janvier Labs (F-53941 Le Genest Saint Isle).
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 week old
- Weight at study initiation:
GROUP 1: 20.3g (Average)
GROUP 2: 20.5g (Average)
GROUP 3: 20.6g (Average)
GROUP 4: 21.5g (Average)

- Housing:individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum):tap water from public distribution system (ad libitum)
- Water (ad libitum):ENVIGO 2016 (ad libitum). It consists of :macronutrients, minerals, amino acids, vitamins, fatty acid and cholesterol.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: 5 days
- Indication of any skin lesions: no skin lesions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-25
- Humidity (%):30-70
- Air changes (per hr):12 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light/12 hours dark
- IN-LIFE DATES: From: To: 16.11.2016-22.11.2016 (6 days)

Vehicle:

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

Remarks:

it produced the most suitable formulation at the required concentration

Concentration:

100%, 50% and 25% of AOO

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
The preliminary test was carried out as the information about the potentional irritating and systemic toxicity of teh test item was not available. Test was performed using one mouse.
- Compound solubility: The 25 μL of the test item undiluted (100%) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).The mouse was observed daily from day 1.
- Irritation:NO
- Systemic toxicity: NO
- Ear thickness measurements:on day 1, day 3 and on day 6
- Erythema scores:NO
- The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and Day 6.
No cutaneous reaction was noted at the tested concentration of 100%.
The 100% concentration was chosen as the highest concentration for the main study.

MAIN STUDY:
Based on the results from preliminary study, the main study was conducted.
- Clinical observations and mortality: All animals were observed daily on days 1,2,3,4,5 and 6. Any sign of toxicity or sign of ill health during test were recorded.
- Body weight: - Body weight of each animal was recorded on day 1 (prior tu testing) and on day 6 (prior tu termination)
- Ear thickness measurements and recording of local reactions
These measuremnts were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses. On day 1 and 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 weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Skin sensititation, LLNA: BrdU ELISA
- Criteria used to consider a positive response: Test item with Stimulation index SI >= 1.6 will be classified as a "sensitiser".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 (i.e. SI value between 1.6 and 1.9) is declared positive.
The positive test item will be classified in subcategory 1A or 1B in accordance with the following table:

SUB-CATEGORY 1A EC value <=2%
SUB-CATEGORY 1B EC value > 2%

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item undiluted (100%) and diluted at 50% and 25% in Acetone/olive oil (4:1, v/v). 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). 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. The concentrations of the test and reference items are indicated in following:

1 group 4 females Vehicle 0%
2 group 4 females test item 25%
3 group 4 females test item 50%
4 group 4 females test item 100%

One additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred in groups during the collecting of lymph nodes, the lymph nodes of the additional mouse were not collected and the data concerning this animal was not used in the study.

BrdU injection:
On day 5,0 .5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised. The Brdu solution was prepared by weighing 251.2 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBD6482V) in a glass sample bottle and adding 25.12 mL of physiological saline. Then, the preparation was magnetically stirred, just before the treatment.

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 leastin 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, 30μL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690nm was then measured.

Preparation of Single Cell Suspension:
From each mouse, a single-cell suspension 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 DPBS (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.

Positive control substance(s):

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

Positive control results:

The substance a-Hexylcinnamaldehyde in accordance with the Regulation (EC) No. 1272/2008 has to be classified in category 1 “Skin sensitisation”. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Key result

Parameter:

SI

Value:

ca. 0.81

Variability:

SD:± 0.11

Test group / Remarks:

Group 2 (25% test item)

Remarks on result:

other: No skin sensititaion

Key result

Parameter:

SI

Value:

ca. 1.01

Variability:

SD:± 0.21

Test group / Remarks:

Group 3 (50% test item)

Remarks on result:

other: No skin sensititation

Key result

Parameter:

SI

Value:

ca. 1.08

Variability:

SD:± 0.26

Test group / Remarks:

Group 4 (100% test item)

Remarks on result:

other: No skin sensititation

Parameter:

other: EC 1.6

Remarks on result:

not determinable

Remarks:

(SI < 1.6)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
EC 1.6: not possible to be measured.(See "Results table")

DETAILS ON STIMULATION INDEX CALCULATION:(measurement explained in "Deatils on Study Desing"). Stimulation index was not more than 1.6 at tested concentration of test item.
The BrdU labelling index was defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
em = emission wavelength; and ref = reference wavelength.

EC1.6 CALCULATION
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) is determined 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

Because of SI not more than 1.6, the EC value can not be determinated.

CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. See Table 2

BODY WEIGHTS: Changes in body weight of tested animals between day 1 and 6 were comparable to those observed in the corresponding control group animals over the same period.

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS: The calculated SI of individual group was following: 25% (0.81), 50% (1.01), 100%(1.08).

LOCAL IRRITATION: No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%.

Table No.1: Main study: Individual clinical observation and mortality data

Group Test item Animals Day 1 Day 2 Day 3 Day 4 Day 5 Day 6   1   AOO Sf8890 0 0 0 0 0 0 Sf8891 0 0 0 0 0 0 Sf8892 0 0 0 0 0 0 Sf8893 0 0 0 0 0 0   2   25% Sf8895 0 0 0 0 0 0 Sf8896 0 0 0 0 0 0 Sf8897 0 0 0 0 0 0 Sf8898 0 0 0 0 0 0   3   50% Sf8900 0 0 0 0 0 0 Sf8901 0 0 0 0 0 0 Sf8902 0 0 0 0 0 0 Sf8903 0 0 0 0 0 0   4   100% Sf8905 0 0 0 0 0 0 Sf8906 0 0 0 0 0 0 Sf8907 0 0 0 0 0 0 Sf8908 0 0 0 0 0 0  0: no sign of systemic toxicity AOO-Acetone/olive oil (4:1,v/v)

Table No.2: Main study: Individual body weight and body weight gain

Date of 1st application (day 1): 16 November 2016

Groups	Test item	Animals	Bodyweight (g)		Day6 ear thickness (mm)
			Day 1                         Day6
1	AOO	N° Sf 8890	19.6	20.8	1.2
		N° Sf 8891	20.8	21.6	0.8
		N° Sf 8892	21.3	22.8	1.5
		N° Sf 8893	19.5	19.4	-0.1
		MEAN	20.3	21.2	0.9
		Standart deviation	0.9	1.4	0.7
2	25%	N° Sf 8895	20.5	20.5	0.0
		N° Sf 8896	21.1	21.9	0.8
		N° Sf 8897	19.9	19.9	0.0
		N° Sf 8898	20.3	21.5	1.2
		MEAN	20.5	21.0	0.5
		Standart deviation	0.5	0.9	0.6
3	50%	N° Sf 8900	22.1	24.2	2.1
		N° Sf 8901	19.5	20.6	1.1
		N° Sf 8902	20.8	21.0	0.2
		N° Sf 8903	19.8	22.0	2.2
		MEAN	20.6	22.0	1.4
		Standart deviation	1.2	1.6	0.9
4	100%	N° Sf 8905	20.4	21.9	1.5
		N° Sf 8906	20.6	22.1	1.5
		N° Sf 8907	23.2	22.6	-0.6
		N° Sf 8908	21.6	23.2	1.6
		MEAN	21.5	22.5	1.0
		Standart deviation	1.3	0.6	1.1

Table No.3: BrdU index & Stimulation index per group and calculation of EC1.6

Date of 1st application (day 1): 16 November 2016

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	AOO	0.730	n.a	n.a	n.a
2	25%	0.595	0.81 ±0.11	Negative	n.a
3	50%	0.736	1.01 ±0.21	Negative
4	100%	0.787	1.08 ±0.26	Negative

n.a.: non applicable

*: mean: Σindividual value / 4

Table No.4: Main study: BrdU index & Stimulation index (individual data)

Date of 1st application (day 1): 16 November 2016

Test item Animals BrdU Index (DO indiv) BrdU Index (DO mean) BrdU Index  Mean* Stimulation index (SI) (Indiv±SD)   AOO Sf8890 0.713   0.876             0.730   n.a 0.995 0.921 Sf8891 0.618   0.759   n.a 0.773 0.887 Sf8892 0.585   0.695   n.a 0.778 0.723 Sf8893 0.689   0.588   n.a 0.546 0.530 Test item Animals BrdU Index (DO indiv) BrdU Index (DO mean) BrdU Index  Mean* Stimulation index (SI) (Indiv±SD)   25% Sf8895 0.573   0.668             0.595   0.92±0.14 0.661 0.772 Sf8896 0.426   0.549 0.75±0.18 0.684 0.539 Sf8897 0.740   0.656 0.90±0.13 0.679 0.549 Sf8898 0.438   0.505 0.69±0.11 0.593 0.484 Test item Animals BrdU Index (DO indiv) BrdU Index (DO mean) BrdU Index  Mean* Stimulation index (SI) (Indiv±SD)   50% Sf8900 0.529   0.728             0.736   1.00±0.24 0.871 0.786 Sf8901 0.732   0.740 1.01±0.04 0.715 0.774 Sf8902 1.052   0.930 1.27±0.17 0.931 0.807 Sf8903 0.548   0.547 0.75±0.01 0.540 0.554
Test item Animals BrdU Index (DO indiv) BrdU Index (DO mean) BrdU Index  Mean* Stimulation index (SI) (Indiv±SD)   100% Sf8905 0.729   0.909             0.787   1.25±0.23 1.059 0.939 Sf8906 0.841   0.981 1.34±0.064 0.598 1.506 Sf8907 0.709   0.668 0.92 ±0.12 0.728 0.569 Sf8908 0.620   0.589 0.81±0.08 0.623 0.524   *: mean: Σindividual value / 4

Table No.5: Main study: Individual Ear thickness and irritation level

Date of 1st application (day 1): 16 November 2016

Groups	Test item	Animals	Day1  ear thickness (mm)	Day3 ear thickness (mm)	Day6 ear thickness (mm)	Ear thickness increase D3/D1(%)	Ear thickness increase D6/D1(%)
1	AOO	N° Sf 8890	0.20	0.22	0.20	10.0	0.0
		N° Sf 8891	0.21	0.20	0.21	-4.8	0.0
		N° Sf 8892	0.20	0.20	0.19	0.0	-5.0
		N° Sf 8893	0.21	0.20	0.20	-4.8	-4.8
		MEAN	0.21	0.21	0.20	0.1	-2.4
		Standart deviation	0.01	0.01	0.01	7.0	2.8
2	25%	N° Sf 8895	0.20	0.21	0.21	5.5	5.0
		N° Sf 8896	0.21	0.21	0.20	0.0	-4.8
		N° Sf 8897	0.21	0.21	0.20	0.0	-4.8
		N° Sf 8898	0.21	0.21	0.20	0.0	-4.8
		MEAN	0.21	0.21	0.20	1.3	-2.3
		Standart deviation	0.00	0.00	0.00	2.5	4.9
3	50%	N° Sf 8900	0.21	0.20	0.20	-4.8	-4.8
		N° Sf 8901	0.21	0.22	0.22	-4.8	4.8
		N° Sf 8902	0.21	0.22	0.22	-4.8	4.8
		N° Sf 8903	0.22	0.22	0.22	0.0	0.0
		MEAN	0.21	0.22	0.22	-3.6	1.2
		Standart deviation	0.01	0.01	0.22	2.4	4.6
4	100%	N° Sf 8905	0.21	0.22	0.01	-9.5	4.8
		N° Sf 8906	0.22	0.22	0.22	-4.5	0.0
		N° Sf 8907	0.20	0.22	0.22	5.0	10.0
		N° Sf 8908	0.21	0.22	0.22	4.8	4.8
		MEAN	0.21	0.22	0.22	-1.1	4.9
		Standart deviation	0.01	0.00	0.00	7.2	4.1

Table No.6: Main study: Individual Ear biopsy weight and lymph node weight

Date of 1st application (day 1): 16 November 2016

Groups	Test item	Animals	ear weigh  Day 6 (mg)	% of ear weight increased/group1	lymph nodes (mg)
1	AOO	N° Sf 8890	27.8		5.0
		N° Sf 8891	27.7		5.5
		N° Sf 8892	28.3		5.1
		N° Sf 8893	27.3		4.9
		MEAN	27.8		5.1
		Standart deviation	0.4		0.3
2	25%	N° Sf 8895	29.1	6.8	5.2
		N° Sf 8896	27.9		6.4
		N° Sf 8897	32.0		8.4
		N° Sf 8898	29.7		5.4
		MEAN	29.7		6.4
		Standart deviation	1.7		1.5
3	50%	N° Sf 8900	30.0	5.9	7.0
		N° Sf 8901	30.2		6.8
		N° Sf 8902	28.4		5.9
		N° Sf 8903	29.1		6.4
		MEAN	29.4		6.5
		Standart deviation	0.8		0.5
4	100%	N° Sf 8905	28.1	3.1	6.0
		N° Sf 8906	29.8		6.6
		N° Sf 8907	28.2		7.9
		N° Sf 8908	28.4		9.2
		MEAN	28.6		7.4
		Standart deviation	0.8		1.4

Table No.7: Main study: Summary of result

Date of 1st application (day 1): 16 November 2016

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation #
1	AOO	-2.4	n.a	NO
2	25%	-2.3	6.8	NO
3	50%	1.2	5.9	NO
4	100%	4.9	3.1	NO

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

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

Interpretation of results:

GHS criteria not met

Conclusions:

The stimulation index of the test item is 0.81, 1.01 and 1.08 at the concentration of 25%, 50% and 100% respectively, on a LLNA study in mouse. As the value of stimulation index is lower than 1.6, the test item is not a sensitizer.

Executive summary:

In accordance with OECD Test Guideline No.442-B, GLP study, this study was to performed to determine the skin sensitization of the test item in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. Three groups of four animals (group 2,3,4) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item of the test item undiluted (100%) and diluted at concentrations of 25% and 50% in Acetone/olive oil (4:1, v/v) (AOO). A group (1) of four animals was treated with vehicle AOO alone. On D5, 0.5 mL of BrdU solution (10 mg/mL) was injected by the intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU concent in DNA of lymphocyte using an ELISA kit. The values obtained were used to calculate stimulation indices (SI). No mortality and no signs of systemic toxicity were noted.

No increase in ear thickness and in ear weight was noted in animals treated at 100%, 50% and 25% - therefore test item is not excessively irritant at these concentrations. The stimulation index was calculated by individual approach : 0.81, 1.01, 1.08 for the treated groups at 25%, 50% and 100%. The stimulation index of every tested group was < 1.6 therefore the test item is not considered as skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The study does not have to be conducted because of already existing in vivo study.

Reason / purpose for cross-reference:

reference to other study

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation in vivo:

Key study: In accordance with OECD Test Guideline No.442-B, GLP study, this study was to determine the skin sensitization of the test item in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. Three groups of four animals (group 2,3,4) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item of the test item undiluted (100%) and diluted at concentrations of 25% and 50% in Acetone/olive oil (4:1, v/v) (AOO). A group (1) of four animals was treated with AOO. Treated animals were checked for clinical signs, body weights, signs of toxicity and mortality, ear thickness. Stimulation index (SI) was calculated for evaluation of skin sensitization. No mortality and no signs of systemic toxicity were noted. No increase in ear thickness and in ear weight was noted, therefore test item is not irritant at tested concentrations. The stimulation index was evaluated as 0.81, 1.01, 1.08 for the treated groups at 25%, 50% and 100%. The stimulation index of every tested group was < 1.6 therefore the test item is not consideredas a skin sensitizer.

Data waiving: In vitro study is not necessary because the study in vivo (LLNA) is already available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, where the evaluated stimulation index was 0.81, 1.01 and 1.08 (less than 1.6) at tested concentrations respectively, the test item is not classified as skin sensitizer according to CLP Regulation (EC) no.1272/2008.