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Description of key information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaCrl
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Animal strain: mouse; CBA/CaCrl (Pre-test: CBA/CaOlaHsd)
- Source: Charles River, UK (Pre-test: Harlan Winkelmann GmbH, Germany)
- Age at study initiation: 8 - 11 weeks
- Mean weight at study initiation: ca. 16.2 - 20.5 g
- Housing: 5/cage; Makrolon cages, type II/III
- Diet: Pelleted standard diet, Harlan Laboratories B.V., Horst, Netherlands; ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 35- 65
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50%, 25% and 10%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 100%. The dilutions were formulated in acetone/olive oil/4:1 (v/v) (AOO).
To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50% (w/w) and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value V3 was observed at any observation time and/or if an increase in ear thickness of V25% was recorded on day 3 or day 6. The measured ear weight was also considered in this evaluation.
At the tested concentrations the animals did not show any signs of systemic toxicity. On day 4 and day 5, the animal treated with the undiluted test item showed an erythema of the ear skin (Score 1). Other signs of irritation or signs of systemic toxicity were not observed.
Additionally, at both tested concentrations, an increase in ear weight was observed that exceeded the threshold value of 25% for excessive local skin irritation mentioned in OECD guideline 429. Measurement of ear thickness did not confirm the ear weight, but was increased at 100% (+ 18.9%). Thus, the test item in the main study was assayed at 10, 25, and 50% (w/w).

MAIN STUDY
TREATMENT SCHEME
Vehicle control group 1: AOO
Test group 2: 10% test item in AOO
Test group 3: 25% test item in AOO
Test group 4: 50% test item in AOO

EXPERIMENTAL PROCEDURE
- Route of application: Epicutaneously to the dorsum of both ears
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Administration of 3H-Methyl Thymidine: Five days after the first topical application 250 µL of phosphate-buffered saline (PBS) containing 20.2 µCi of 3HTdR (equivalent to approximately 80.9 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
- Determination of Incorporated 3HTdR: Approximately five hours after treatment with 3HTdR all mice were sacrificed. The draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared. The level of 3HTdR incorporation was measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 63110 Rodgau, Germany). 3HTdR incorporation was expressed as the number of radioactive disintegrations per minute (DPM).
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal using a cell counter.
- Determination of ear weight: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch. For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.
- Evaluation of results: The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.).

CLINICAL EXAMINATIONS
- Body weight determination: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: after the first application and prior to treatment with 3HTdR.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were checked individually.
- Mortality: A check for moribund and dead animals was made at least once each workday.

CALCULATION OF RESULTS
- Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean 3HTdR labelling index/mouse within each test substance group by the mean 3HTdR labelling index for the negative control (NC) group. The average SI for the NCs is then one.
- A result is regarded as positive when SI ≥ 1.55.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Calculation of mean and standard deviation was performed.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test group 2 (10%): mean: 3.58 (individual values: 2.6, 5.1, 4.6, 2.0, and 3.6) Test group 3: (25%): mean: 9.94 (individual values: 7.5, 11.9, 10.4, 11.5, and 8.5) Test group 4: (50%): mean: 9.83 (individual values: 6.9, 10.5, 11.0, 10.4, and 10.4) SI-value of historical positive control: 8.08 at 25% alpha hexylcinnamaldehyde
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Test group 1 (vehicle control; NC): mean: 1127.8 (individual values: 1317, 1594, 1038, 741, and 1059) Test group 2 (10%): mean: 4041.8 (individual values: 2988, 5750, 5192, 2301, and 4088) Test group 3 (25%): mean: 11209.2 (individual values: 8462, 13396, 11719, 13026, and 9553) Test group 4 (50%): mean: 11081.0 (individual values: 7792, 11892, 12424, 11711, and 11696)

Table 1 Lymph Node Cell Count

Test item concentration
% (w/w)

Group Calculation

Mean lymph node

cell count (x10E06 per group)

Standard deviation

Index

Vehicle
(AOO)

9.78

1.93

1.0

10 %
test substance

19.28*

5.81

1.97

25 %
test substance

31.83*

4.92

3.25

50 %
test substance

33.42*

5.69

3.42

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Table 2 Lymph Node Weight

Test item concentration
% (w/w)

Group Calculation

Mean Lymph Node Weight (mg per group)

Standard deviation

Index

Vehicle
(AOO)

6.15

0.79

1.0

10 %
test substance

9.29*

1.17

1.51

25 %
test substance

13.11*

1.58

2.13

50 %
test substance

13.90*

2.01

2.26

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Table 3 Ear Weights

Test item concentration
% (w/w)

Group Calculation

Mean ear weight (mg per group)

Standard deviation

Index

Vehicle
(AOO)

24.89

3.40

1.0

10 %
test substance

27.46

2.38

1.10

25 %
test substance

34.29*

4.08

1.38

50 %
test substance

47.76*

9.71

1.92

Index = values of the test item groups related to the mean value of the control group

* statistically significant increase in comparison to vehicle control group (p ≤ 0.05)

 

Further observations:

No deaths occurred during the study period. No systemic findings were observed during the study period. On day 4 and day 5, the animals treated with 25 and 50% of the test item showed an erythema of the ear skin (score 1). The animals treated with 50% test item additionally showed an erythema of the ear skin (score 1) on day 6. Animals treated with 10% test item did not show any sign of local skin irritation.

The body weight of the animals was within the range commonly recorded for animals of this strain and age.  

Interpretation of results:
sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

There are valid data available for the assessment of the sensitising potential.

LLNA

In the key study the skin sensitising property of the test substance was determined using the LLNA (Harlan, 1445505 / BASF SE 58V0587/11 X 160, 2012). The study was conducted under GLP according to the OECD-Guideline 429 based on the use of in vivo radioactive labelling (³HTdR) to measure an increased number of proliferating cells in the draining auricular lymph nodes. The test substance was dissolved in acetone/olive oil (4:1 v/v) at concentrations of 50, 25 and 10%, and 25 µL/ear/day were topically administered to 5 female CBA/CaCrl mice each dose group once daily on 3 consecutive days. Additionally, a control group was treated with the vehicle alone. The sensitivity of the strain was assured in August 2011 by testing α-hexyl cinnamaldehyde as positive control.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 4 and day 5, the animals treated with the test item concentrations of 25 and 50% showed an erythema of the ear skin (Score 1). The animals treated with 50% test item concentration additionally showed an erythema of the ear skin (Score 1) on day 6. Animals treated with 10% test item concentration did not show any signs of local skin irritation. A statistically significant and biologically relevant increase in ear weights was observed in the mid and the high dose group in comparison to the vehicle control group (p<0.05). An increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was exceeded in the mid and high dose group (37.8 and 91.9%, respectively), thus indicating the irritant properties of the test item in the mid and high dose group, but not in the low dose group.

Stimulation Indices (SI) were calculated and revealed values of 3.58, 9.94, and 9.83 for the test item concentrations of 10, 25, and 50% (w/w). The EC3 value could not be calculated, since all S.I.s are above the threshold of 3. A statistically significant and biological relevant increase in DPM value and also in lymph node weight and cell count was observed in all dose groups in comparison to the vehicle control group.

The study is suitable and reliable for assessment of skin sensitisation as it was performed according to OECD test guideline.

Based on the above mentioned findings regarding ear skin irritation, an influence of irritation on lymphocyte proliferation cannot be excluded, but only in the mid and high dose group. Nevertheless, on the basis of the present data, the test item has to be classified as a sensitiser.


Migrated from Short description of key information:
Mouse local lymphnode assay (LLNA): sensitising (Val 1, GLP, OECD 429, mouse, Harlan 1445505, 2012)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data for sensitisation are reliable and suitable for the purpose of classification under Directive 67/548/EEC. As a result the substance is considered to be classified for sensitisation (Xi, R43) under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data for sensitisation are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. As a result the substance is considered to be classified for sensitisation (Cat 1B, H317, May cause an allergic skin reaction) under Regulation (EC) No.1272/2008.