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Description of key information

Skin sensitisation: Key study. Method according to OECD 422B, GLP study. The Stimulation Index (SI) was 1.50, 1.43, 1.32 for the treated groups at 50%, 25% and 10%, respectively. Therefore, the test item has no sensitisation potential.

Data waiving (study scientifically not necessary / other information available): data from a LLNA study according to OECD 422B was available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 23rd to December 12th, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Test material information:
Composition 1
Species:
mouse
Strain:
CBA:J
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: 8 or 9 weeks old.
- Weight at study initiation: average weight 21.2 g (SD: 1.1)
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): 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).
- Water (e.g. ad libitum): Tkelad Global 18% Protein Rodent Diet (ENVIGO 2016) ad libitum.
- Acclimation period: at least 5 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30% to 70%
- Air changes (per hr): at least 10.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light.
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: See Table 1 in 'Any other information on materials and methods incl. tables'. Due to the physical characteristics of the test item, the formulation at 50 % (w/v) using DMF as vehicle was suitable for the test (highest achievable concentration).
- Irritation: no.
- Systemic toxicity: no.
- Ear thickness measurements: values were within the acceptable range, see 'Any other information on materials and methods incl. tables'.
- Erythema scores: no signs of erythema were observed. See 'Any other information on materials and methods incl. tables'.

MAIN STUDY
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in DMF, 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 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: 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, 30µL 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 SI value was derived as specified in the guidelines. If at least one concentration of the test item results is greater than 1.6 compared to control values, that is considered a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in DMF, 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). 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 (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.
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. Then, BrdU was determined.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The results for the last positive control (29/06/2016) were within the acceptable ranges. 5%, 10% and 25% solutions of hexyl cinnamic aldehyde in MEK generated a SI = 1.34, 1.63 and 1.95, respectively. The EC1.6 value was 9.48%.
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.43
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
10%
Parameter:
other: EC1.6
Remarks on result:
not determinable
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: see 'Any other information on results' below.

DETAILS ON STIMULATION INDEX CALCULATION: as recommended by the guideline. No stimulation index of more than 1.6 was recorded whatever the tested concentration.

EC CALCULATION: 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), where 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.

CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Depilation behind right ear was noted in two animals and depilation behind two ears was noted in the two other on day 3. Depilation was noted behind two ear in all treated animals with test item diluted at 50% between days 4 and 6. 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.

Table 3. Main study: Individual clinical observation and mortality data.

Groups

Animals

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Vehicle (DMF)

Sf 9084

0

0

0

0

0

0

Sf 9085

0

0

0

0

0

0

Sf 9086

0

0

0

0

0

0

Sf 9087

0

0

0

0

0

0

10% test item

Sf 9089

0

0

0

0

0

0

Sf 9090

0

0

0

0

0

0

Sf 9091

0

0

0

0

0

0

Sf 9092

0

0

0

0

0

0

25% test item

Sf 9094

0

0

0

0

0

0

Sf 9095

0

0

0

0

0

0

Sf 9096

0

0

0

0

0

0

Sf 9097

0

0

0

0

0

0

50% test item

Sf 9099

0

0

0 -

0 -

0-

Sf 9100

0

0

0 -

0 -

0 -

Sf 9101

0

0

0 -

0 -

0 -

0 -

Sf 9102

0

0

0-

0 -

0 -

0 -

0: no sign of systemic toxicity; MEK: methyl ethyl ketone; (º): residue of test item; (*): slight dryness; (**): dryness; (***): scab.

Table 4. Main study: Individual body weight and body weight gain.

Groups

Animals

Body weight

(g)

Body weight gain

(g)

Day 1

Day 6

Vehicle

(DMF)

Sf 9084

20.9

21.6

0.7

Sf 9085

20.1

20.6

0.5

Sf 9086

20.2

21.6

1.4

Sf 9087

21.4

22.8

1.4

Mean

20.7

21.7

1.0

SD

0.6

0.9

0.5

10% test item

Sf 9089

23.6

23.9

0.3

Sf 9090

20.8

21.5

0.7

Sf 9091

20.9

21.5

0.6

Sf 9092

20.2

20.5

0.3

Mean

21.4

21.9

0.5

SD

1.5

1.4

0.2

25% test item

Sf 9094

20.0

19.3

-0.7

Sf 9095

22.2

21.9

-0.3

Sf 9096

21.0

21.7

0.7

Sf 9097

20.6

20.9

0.3

Mean

21.0

21.0

0.0

SD

0.9

1.2

0.6

50% test item

Sf 9099

20.3

19.4

-0.9

Sf 9100

22.4

22.9

0.5

Sf 9101

22.4

22.0

-0.4

Sf 9102

23.0

22.3

-0.7

Mean

22.0

21.7

-0.4

SD

1.2

1.5

0.6

 

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

Groups

BrdU-index (mean)

SI (mean + SD)

Result

EC1.6

Vehicle (DMF)

0.411

n.a.

n.a.

n.a.

10% test item

0.544

1.32 ± 0.10

Negative

n.a.

25% test item

0.587

1.43 ± 0.15

Negative

50% test item

0.615

1.50 ± 0.13

Negative

n.a.: not applicable; MEK: methyl ethyl ketone.

Table 6. Summary of results – skin irritation.

Groups

Ear thickness increase

D6/D1 (%)

Biopsy ear

weight increase

(%)

Excessive .

irritation #

Vehicle (DMF)

4.3

n.a.

No

10% test item

2.4

0.7

No

25% test item

-2.4

-2.1

No

50% test item

-3.6

4.2

No

Table 7. Individual Ear thickness and irritation level.

Groups

Animals

Day 1

(mm)

Day 3

(mm)

Day 6

(mm)

Ear thickness increase

D3/D1 (%)

Ear thickness increase

D3/D1 (%)

Vehicle (DMF)

Sf 9084

0.19

0.21

0.22

10.5

15.8

Sf 9085

0.19

0.21

0.21

10.5

10.5

Sf 9086

0.22

0.20

0.20

-9.1

-9.1

Sf 9087

0.21

0.20

0.21

-4.8

0.0

Mean

0.20

0.21

0.21

1.8

4.3

SD

0.02

0.01

0.01

10.2

11.1

10% test item

Sf 9089

0.21

0.21

0.22

0.0

4.8

Sf 9090

0.20

0.20

0.21

0.0

5.0

Sf 9091

0.21

0.19

0.22

-9.5

4.8

Sf 9092

0.21

0.19

0.20

-9.5

-4.8

Mean

0.21

0.20

0.21

-4.8

2.4

SD

0.00

0.01

0.01

5.5

4.8

25% test item

Sf 9094

0.21

0.21

0.20

0.0

-4.8

Sf 9095

0.21

0.20

0.20

-4.8

-4.8

Sf 9096

0.21

0.22

0.21

4.8

0.0

Sf 9097

0.21

0.22

0.21

4.8

0.0

Mean

0.21

0.21

0.21

1.2

-2.4

SD

0.00

0.01

0.01

4.6

2.7

50% test item

Sf 9099

0.19

0.19

0.19

0.0

0.0

Sf 9100

0.21

0.19

0.20

-9.5

-4.8

Sf 9101

0.21

0.20

0.20

-4.8

-4.8

Sf 9102

0.21

0.20

0.20

-4.8

-4.8

Mean

0.21

0.20

0.20

-4.8

-3.6

SD

0.01

0.01

0.01

3.9

2.4

 

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

Groups

Animals

Ear weight

Day 6

(mg)

% ear weight

increased/group 1

Lymph

nodes

(mg)

Vehicle (MEK)

Sf 9084

27.3

 -

4.6

Sf 9085

27.8

4.4

Sf 9086

27.8

4.7

Sf 9087

28.1

4.6

Mean

27.8

4.6

SD

0.3

0.1

10% test item

Sf 9089

28.9

0.7

5.1

Sf 9090

27.0

5.2

Sf 9091

27.5

5.2

Sf 9092

28.4

5.0

Mean

28.0

5.1

SD

0.9

0.1

25% test item

Sf 9094

26.0

-2.1

5.4

Sf 9095

26.9

5.0

Sf 9096

27.7

5.3

Sf 9097

28.1

5.8

Mean

27.2

5.4

SD

0.9

0.3

50% test item

Sf 9099

29.2

4.2

6.1

Sf 9100

27.8

6.5

Sf 9101

29.6

6.8

Sf 9102

29.1

7.0

Mean

28.9

6.6

SD

0.8

0.4

 

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
The test item did not show skin sensitisation potential under the tested conditions in the LLNA assay. The Stimulation Indexes were 1.50, 1.43, 1.32 at concentrations of 50, 25 and 10% test item in DMF, respectively. Therefore, the test item is not sensitising.
Executive summary:

The skin sensitisation potential of the test item was studied according to OECD 422B, under GLP conditions. Four groups of four female CBA/J mice received 25 µL/ear of 50, 25, and 10% (w/v) test item in dimethylformamide; and one negative control group received the vehicle (DMF). The last positive control results were used for reference. The animals were treated for 3 consecutive days (D1, D2, D3), and injected with BrdU solution on Day 5. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content 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 in the test and control animals during the test. . Depilation behind right ear was noted in two animals and depilation behind two ears was noted in the two other on day 3. Depilation was noted behind two ear in all treated animals with test item diluted at 50% between days 4 and 6. 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 1.50, 1.43, 1.32 for the treated groups at 50%, 25% and 10%, respectively. In conclusion, under the conditions of the present assay, the test item was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. Based on the results, the test item does not need classification according to GHS or CLP criteria.

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
In accordance with REACH Annex XI, the study does not appear scientifically necessary, as a skin sensitisation in vivo study (LLNA) was available (1.1. Use of existing data).
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Key study. Method according to OECD 422B, GLP study. Four groups of four female CBA/J mice received 25 µL/ear of 50, 25, and 10% (w/v) test item in dimethylformamide; and one negative control group received the vehicle (DMF). The last positive control results were used for reference. The animals were treated for 3 consecutive days (D1, D2, D3), and injected with BrdU solution on Day 5. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. The values obtained were used to calculate stimulation indices (SI). Depilation behind right ear was noted in two animals and depilation behind two ears was noted in the two other on day 3. Depilation was noted behind two ear in all treated animals with test item diluted at 50% between days 4 and 6. 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 was1.50, 1.43, 1.32 for the treated groups at 50%, 25% and 10%, respectively. In conclusion, under the conditions of the present assay, the test item had no sensitisation potential.

Data waiving (study scientifically not necessary / other information available): 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 (SI < 1.6 in OECD 442B test), the substance is not classified for sensitising properties in accordance with CLP Regulation (EU) No. 1272/2008.