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Description of key information

The substance was found to be skin sensitising when tested in the murine Local Lymph Node Assay according to OECD 429 guideline.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 november 2015 - 14 december 2015
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
july 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: the animals of the preliminary test were approximately 10-12 weeks and the animals of the main test were approximately 10 weeks old
- Mean body weight at study initiation: the animals of the preliminary test and the main test had a bodyweight within +/- 20% of the sex mean.
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-Z pelleted diet (free access)
- Water: yes (free access)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr):at least 10 cycles/hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Acetone p.a.: Merck, Darmstadt, Germany / olive oil: Fagron, Nieuwerkerka/d Ijssel, The Netherlands
Concentration:
For the preliminary test the concentrations were 50 and 100%, of the test item.
For the main test the concentrations were 0,10, 50 and 100% of the test item.
No. of animals per dose:
For the preliminary test: 2 females/dose (no controls)
For the main test: 5 females/dose, 5 females for the negative control and 5 females for the positive control
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility using test item data and trial preparation results: the first recommended vehicle (acetone/olive oil) was chosen as a homogenous dosage form preparation was obtained at the concentration of 100% inacetone/olive oil (4:1 v/v).
- Irritation: In the preliminary test, measurement of the ear thickness (using a digital thickness gauge) was performed prior to dosing on Days 1 and 3, and on Day 6. Increase in ear thickness was below 25% in all animals, the maximum value being +7%. In the preliminary test, skin irritation grade 1 or 2 was noted in all females treated at 50% and 100%. The highest concentration retained for the main test was therefore 100%.
- Lymph node proliferation response:not assessed


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: Disintegration per minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI = 3, the test item may be regarded as a skin sensitizer. The EC3 values (the estimated test item concentration that will give a SI =3) were determined, using
linear interpolation.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was prepared in the vehicule at the chosen concentrations. All dosage form preparations were prepared within 4 hours prior to each dosing. On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None
Positive control results:
SI =4.6 was obtained for the positive control (i.e. HCA 25% in AOO). The experiment was therefore considered valid.
Key result
Parameter:
EC3
Value:
58.1
Key result
Parameter:
SI
Remarks on result:
other: The SI values calculated for the test item concentrations 10, 50 and 100% were 1.5; 2.4 and 6.1 respectively

Table 7.4.1/1: Relative size lymph nodes, Radioactivity counts (DPM) and Stimulation Index (SI)

Group

Test Item (%)

Animal

Size nodes

 

Left               Right

DPM/animal

Mean

DPM±SEM

Mean

SI±SEM

1

Vehicle control

1

n

n

515

996± 214

1.0± 0.3

2

n

n

671

3

n

n

779

4

n

n

1437

5

n

n

1576

2

10%

6

n

n

1385

1457± 158

1.5± 0.4

7

n

n

1100

8

n

n

2039

9

n

n

1478

3

50%

10

n

n

1284

2365± 391

2.4± 0.6

11

n

n

2118

12

n

n

1535

13

n

n

2280

14

n

n

2044

4

100%

15

+

+

3846

6047± 577

6.1± 1.4

16

+

+

7052

17

+

+

5737

18

+

+

6735

19

+

+

6790

20

+

n

3920

11

Positive control

51

n

n

4599

4600± 399

4.6± 1.1

52

n

n

4000

53

+

+

6136

54

+

+

4250

55

+

+

4015

Test Item (% w/w)

Relative size auricular lymph nodes: -, --, ---: degree of reduction, +,++,+++: degree of enlargment, n: considered to be normal

DPM= Disintegrations per minute

SEM= Standard Error of the Mean

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item induces delayed contact hypersensitivity in the murine Local Lymph Node Assay ( EC3 = 58,1%)
Executive summary:

The objective of this study was to evaluate the potential of the test substance to induce contact hypersensitivity in CBA female mice using the murine Local Lymph Node Assay (LLNA).

This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices.

To assess the irritant potential of the test substance, a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test substance diluted in in acetone/olive oil (4:1 v/v) by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Increase in ear thickness was below 25% in all animals, the maximum value being +7%. Skin irritation grade 1 or 2 was noted in all females treated at 50% and 100%. The highest concentration retained for the main test was therefore 100%.

In the main test, three groups of five female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 10, 50 or 100% under a dose-volume of 25 µL. One negative control group of five females received the vehicle (acetone/olive oil (4:1 v/v)) under the same experimental conditions. Additionally, one positive control group of five females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions. Once daily on days 1 to 6 and within 1 hour after dosing on days 1 to 3, the local reactions were recorded.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph nodes draining the application site was measured by incorporation of3H-TdR.The results are expressed as disintegrations per minute (dpm) per animal and for each dose group. The obtained values were used to calculate Stimulation Indices (SI). The EC3 value was determined using linear interpolation.

No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the study. Body weight of animals was unaffected by the test substance treatment. The very slight irritation (grade 1) noted on days 2 and /or 3, on days 1 to 3 and on days 1 to 5 for animals treated at 10, 50 and 100% respectively and scaliness of the ears for all females treated at 100% on days 5 and / or 6 were considered not to have a toxicologically significant effect on the activity of the nodes. The auricular lymph nodes of the animals treated at 10 and 50% were considered normal in size while the nodes of animals treated at 100% were considered enlarged. Mean DPM / animal values were 1457, 2365 and 6047 DPM for the 10, 50 and 100% treated groups respectively.The corresponding SI values were 1.5, 2.4 and 6.1. The test item showed a clear dose-response with an EC3 value of 58.1%. The SI value of the positive control was 4.6; this experiment was therefore considered valid.

It was concluded that the test item induced delayed contact hypersensitivity in the murine Local Lymph Node.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practices.

To assess the irritant potential of the test substance, a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test substance diluted in in acetone/olive oil (4:1 v/v) by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Increase in ear thickness was below 25% in all animals, the maximum value being +7%. Skin irritation grade 1 or 2 was noted in all females treated at 50% and 100%. The highest concentration retained for the main test was therefore 100%.

In the main test, three groups of five female mice received the test item by topical route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 10, 50 or 100% under a dose-volume of 25 µL. One negative control group of five females received the vehicle (acetone/olive oil (4:1 v/v)) under the same experimental conditions. Additionally, one positive control group of five females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions. Once daily on days 1 to 6 and within 1 hour after dosing on days 1 to 3, the local reactions were recorded.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph nodes draining the application site was measured by incorporation of3H-TdR. The results are expressed as disintegrations per minute (dpm) per animal and for each dose group. The obtained values were used to calculate Stimulation Indices (SI). The EC3 value was determined using linear interpolation.

No unscheduled deaths and no clinical signs indicative of systemic toxicity were observed during the study. Body weight of animals was unaffected by the test substance treatment. The very slight irritation (grade 1) noted on days 2 and /or 3, on days 1 to 3 and on days 1 to 5 for animals treated at 10, 50 and 100% respectively and scaliness of the ears for all females treated at 100% on days 5 and / or 6 were considered not to have a toxicologically significant effect on the activity of the nodes. The auricular lymph nodes of the animals treated at 10 and 50% were considered normal in size while the nodes of animals treated at 100% were considered enlarged. Mean DPM / animal values were 1457, 2365 and 6047 DPM for the 10, 50 and 100% treated groups respectively. The corresponding SI values were 1.5, 2.4 and 6.1. The test item showed a clear dose-response with an EC3 value of 58.1%. The SI value of the positive control was 4.6; this experiment was therefore considered valid.

It was concluded that the test item induced delayed contact hypersensitivity in the murine Local Lymph Node.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

The substance induced delayed contact hypersensitivity in the murine Local Lymph Node. According to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, the substance is classified Skin sensitizer category 1B as the EC3 value is higher than 2%.

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