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Toxicological information

Skin sensitisation

Currently viewing:

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: EU method B.51 (Skin Sensitization: LLNA: BrdU-ELISA)
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 diphenylcarbamate
EC Number:
210-047-0
EC Name:
Ethyl diphenylcarbamate
Cas Number:
603-52-1
Molecular formula:
C15H15NO2
IUPAC Name:
ethyl N,N-diphenylcarbamate
Test material form:
solid: particulate/powder

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 nulliparous and non-pregnant: yes.
- Age at study initiation: 9 weeks old.
- Weight at study initiation: weight at day 1: 19,7 - 28,2 g
- Housing: The animals were individully housed (to avoid ingestion of test item or the licking of the ear) in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: ENVIGO 2016 supplied ad libitum.
- Water: tap water from public distribution system supplied 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 five days under stabling and nutritional conditions identical to those of the test.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): 10 changes per hour.
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
- IN-LIFE DATES: From: November 16, 2016 To: November 21, 2016

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
50%, 25% and 10%.
The top dose was chosen as it produced the most suitable formulation with the vehicle (see "Any other information on materials and methods incl. tables").
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 dimethyl sulfoxide 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 dimethyl sulfoxide
- Irritation: No cutaneous reaction was noted at the tested concentration.
- Systemic toxicity: No mortality and no sign of systemic toxicity were noted.
- Ear thickness measurements: Values were in the acceptable range (Table 2).
- Erythema scores: no signs of erythema were observed (Table 2).

MAIN STUDY
Groups of four mice were treated with the test item diluted at 50%, 25% adn 10% in dimethyl sulphoxid (DMSO) based on the results of the pre-screen test. 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).
- Clinical observations and mortality: 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 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 sensitisation, Local Lymph Node Assay: BrdU-ELISA
- Criteria used to consider a positive response: 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.
- Criteria used to consider a positive response: The SI value was derived as specified in the guidelines. 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 diluted at 50%, 25% and 10% in dimethyl sulfoxide. 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.
On Day 5, 0.5 mL (5mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. 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.
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 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 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The EC1.6 value was 9.48% (Positive Control Study number: nº LLNA-BrdU-2016-B).
The substance a-Hexylcinnamaldehyde in accordance with the Regulation (EC) No. 1272/2008 has to be classified in category 1 “Skin sensitisation”.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 0.95
Test group / Remarks:
Group 2 (10%)
Key result
Parameter:
SI
Value:
ca. 0.98
Test group / Remarks:
Group 3 (25%)
Key result
Parameter:
SI
Value:
ca. 0.98
Test group / Remarks:
Group 4 (50%)
Key result
Parameter:
other: EC 1.6
Remarks on result:
not determinable
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION: No stimulation index of more than 1.6 was recorded whatever the tested concentration.
Determination of cell proliferation:
Results were expressed as the Stimulation Index (SI).
Results for each treatment group were expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group.

SI = (Mean BrdU labelling index/mouse Treated Group) / (Mean BrdU labelling index/mouse Control Group)

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 (i.e. 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".

Determination of the EC1.6 value: EC 1.6 cannot be determined.
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was 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

CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.
Slight dryness of the skin was noted on day 6 in all treated animals.
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.

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.

Any other information on results incl. tables

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

DMSO

Nº Sf 8870

0

0

0

0

0

0*

Nº Sf 8871

0

0

0

0

0

0*

Nº Sf 8872

0

0

0

0

0

0*

Nº Sf 8873

0

0

0

0

0

0*

2

10%

Nº Sf 8875

0

0

0

0

0

0*

Nº Sf 8876

0

0

0

0

0

0*

Nº Sf 8879

0

0

0

0

0

0*

Nº Sf 8878

0

0

0

0

0

0*

3

25%

Nº Sf 8880

0

0

0

0

0

0*

Nº Sf 8881

0

0

0

0

0

0*

Nº Sf 8882

0

0

0

0

0

0*

Nº Sf 8883

0

0

0

0

0

0*

4

50%

Nº Sf 8885

0

0

0o

0

0

0*

Nº Sf 8886

0

0

0o

0

0

0*

Nº Sf 8887

0

0

0o

0

0

0*

Nº Sf 8888

0

0

0o

0

0

0*

0: No sign of systemic toxicity

DMSO: Dimethyl sulfoxide

*: slight dryness

Table 4. Individual body weight and body weight gain

 

Groups

Test item

Amimals No.

Bodyweight (g)

Bodyweight gain (g)

Day 1

Day 6

1

DMSO

Nº Sf 8870

21.0

22.4

1.4

Nº Sf 8871

25.2

26.2

1.0

Nº Sf 8872

22.8

23.5

0.7

Nº Sf 8873

23.2

23.9

0.7

MEAN

21.3

24.0

0.9

Standard-deviation

1.7

1.6

0.3

2

10%

Nº Sf 9029

19.7

19.8

0.1

Nº Sf 9030

21.3

20.6

-0.7

Nº Sf 9031

22.7

22.8

0.1

Nº Sf 9032

22.4

23.1

0.7

MEAN

21.5

21.6

0.1

Standard-deviation

1.4

1.6

0.6

3

25%

Nº Sf 9034

22.4

23.8

1.4

Nº Sf 9035

28.2

27.1

-1.1

Nº Sf 9036

24.4

24.8

0.4

Nº Sf 9037

23.0

23.5

0.5

MEAN

24.5

24.8

0.3

Standard-deviation

2.6

1.6

1.0

4

50%

Nº Sf 9039

24.0

26.2

2.2

Nº Sf 9040

23.9

25.2

1.3

Nº Sf 9041

22.6

24.3

1.7

Nº Sf 9042

24.3

25.1

0.8

MEAN

23.7

25.2

1.5

Standard-deviation

0.8

0.8

0.6

DMSO: Dimethyl sulfoxide

 

 

 Table 5. BrdU index & Stimulation index per group and calculation of EC1.6

 

Groups

Test item

BrdU-index (mean*)

Stimulation Index SI (mean + standard deviation)

Result

EC1.6 value

1

DMSO

0.950

n.a.

n.a.

n.a.

2

10%

0.902

0.95±0.23

negative

n.a

3

25%

0.934

0.98±0.07

positive

4

50%

0.927

0.98±0.16

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

DMSO

Sf 8870

1.070

1.138

0.950

n.a

1.272

1.072

Sf 8871

0.989

0.965

n.a

0.996

0.909

Sf 8872

0.881

0.888

n.a

0.987

0.796

Sf 8873

0.894

0.809

n.a

0.748

0.785

2

10%

Sf 8875

1.047

0.996

0.902

1.05±0.05

0.972

0.967

Sf 8876

1.064

1.102

1.16±0.07

1.060

1.181

Sf 8879

0.930

0.916

0.96±0.04

0.868

0.948

Sf 8878

0.566

0.592

0.62±0.06

0.653

0.556

3

25%

Sf 8880

1.024

1.027

0.934

1.08±0.07

1.095

0.962

Sf 8881

0.895

0.915

0.96±0.03

0.949

0.901

Sf 8882

0.859

0.882

0.93±0.05

0.848

0.937

Sf 8883

0.960

0.912

0.96±0.07

0.938

0.837

4

50%

Sf 8885

1.123

1.156

0.927

1.22±0.09

1.094

1.250

Sf 8886

0.912

0.866

0.91±0.05

0.815

0.869

Sf 8887

0.870

0.826

0.87±0.04

0.814

0.792

Sf 8888

0.919

0.859

0.90±0.06

0.855

0.802

*: mean:Σindividual value / 4

DMSO: Dimethyl sulfoxide

 

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

DMSO

Nº Sf 8870

0.20

0.22

0.22

10.0

10.0

Nº Sf 8871

0.23

0.22

0.22

-4.3

-4.3

Nº Sf 8872

0.22

0.19

0.22

-13.6

0.0

Nº Sf 8873

0.21

0.22

0.23

4.8

9.5

MEAN

0.22

0.21

0.22

-0.8

3.8

Standard-deviation

0.01

0.02

0.01

10.4

7.1

2

10%

Nº Sf 8875

0.21

0.22

0.21

4.8

0.0

Nº Sf 8876

0.22

0.22

0.20

0.0

-9.1

Nº Sf 8879

0.21

0.22

0.21

4.8

0.0

Nº Sf 8878

0.21

0.21

0.21

0.0

0.0

MEAN

0.21

0.22

0.21

2.4

-2.3

Standard-deviation

0.01

0.01

0.00

2.7

4.5

3

25%

Nº Sf 8880

0.21

0.21

0.21

0.0

0.0

Nº Sf 8881

0.21

0.20

0.22

-4.8

4.8

Nº Sf 8882

0.21

0.21

0.23

0.0

9.5

Nº Sf 8883

0.20

0.20

0.22

0.0

10.0

MEAN

0.21

0.21

0.22

-1.2

6.1

Standard-deviation

0.00

0.01

0.01

2.4

4.7

4

50%

Nº Sf 8885

0.22

0.22

0.23

0.0

4.5

Nº Sf 8886

0.21

0.23

0.23

9.5

9.5

Nº Sf 8887

0.23

0.21

0.23

-8.7

0.0

Nº Sf 8888

0.22

0.21

0.23

-4.5

4.5

MEAN

0.22

0.22

0.23

-0.9

4.5

Standard-deviation

0.01

0.01

0.00

7.8

3.9

DMSO: Dimethyl sulfoxide

 

  

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

DMSO

Nº Sf 8870

31.7

 

6.2

Nº Sf 8871

29.2

6.4

Nº Sf 8872

30.6

6.5

Nº Sf 8873

29.5

6.1

MEAN

30.3

6.3

Standard-deviation

1.1

0.2

2

10%

Nº Sf 8875

27.5

-6.1

6.3

Nº Sf 8876

27.5

9.0

Nº Sf 8879

31.0

6.6

Nº Sf 8878

27.6

6.4

MEAN

28.4

7.1

Standard-deviation

1.7

1.3

3

25%

Nº Sf 8880

27.4

-5.2

6.3

Nº Sf 8881

28.8

6.3

Nº Sf 8882

30.0

6.4

Nº Sf 8883

28.5

6.3

MEAN

28.7

6.3

Standard-deviation

1.1

0.1

4

50%

Nº Sf 8885

29.8

-3.6

8.1

Nº Sf 8886

28.3

6.8

Nº Sf 8887

27.6

7.3

Nº Sf 8888

30.9

6.9

MEAN

29.2

7.3

Standard-deviation

1.5

0.6

DMSO: Dimethyl sulfoxide

 

 

 

Table 9. Summary of result – skin irritation

 

Groups

Test item

Ear thickness increase D6/D1 (%)

Biopsy ear weight Increase (%)

Excessive irritation#

1

DMSO

3.8

n.a

No

2

10%

-2.3

-6.1

No

3

25%

6.1

-5.2

No

4

50%

4.7

-3.6

No

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

DMSO: Dimethyl sulfoxide

 

 

 

Table 10. BrdU index & Stimulation index per group and calculation of EC1.6of 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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item did not show skin sensitisation potential under the tested conditions in the LLNA assay. The Stimulation Index (SI) calculated by individual approach was 0.95, 0.98 and 0.98 for the treated groups at 50%, 25% and 10% in DMSO, respectively. Therefore, EC 1.6 value cannot be determined.
Executive summary:

A local lymph node assay (LLNA:BrdU) was performed according OECD Guideline 442 -B to asses the skin sensitisation potential of the test item in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. The basic principle underlying the LLNA: BrdU is that sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test item application. As the test item is a powder, it was not possible to test the undiluted test item. The highest tested concentration was 50%. Three groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item diluted at concentrations of 50%, 25% and 10% in dimethylsulfoxyde (DMSO). A further group of four animals was treated with DMSO. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Slight dryness of the skin was noted on day 6 in all treated animals.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 0.95, 0.98 and 0.98 for the treated groups at 50%, 25% and 10%, respectively. Therefore, the EC 1.6 cannot be determined. The results obtained, in these experimental conditions, enable to conclude that the test item Ethyl diphenylcarbamate (CAS number 603-52-1) does not have to be classified as a skin sensitizer, in accordance with CLP Regulation EC No. 1272/2008.