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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl ethyl(phenyl)carbamate
EC Number:
213-796-1
EC Name:
Ethyl ethyl(phenyl)carbamate
Cas Number:
1013-75-8
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl ethyl(phenyl)carbamate
Test material form:
liquid
Details on test material:
Colorless
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).

In vivo test system

Test animals

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)

Study design: in vivo (LLNA)

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)

Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
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%.

Any other information on results incl. tables

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)

Applicant's summary and conclusion

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.