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Description of key information

Based on the available information, the test item did not induce a biologically relevant (no increase above the cut off Stimulation Index of 3) or statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, therefore the test item is not considered a skin sensitiser.

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:
08 Nov 2016 to 05 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
6 July 2012
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Test-substance No.: 16/0051-1
- Name of test substance (as reported in the study report): Orasol Black X45
- Batch identification: 002-150504
- Purity: 99.22 area-% (sum of all peaks, HPLC 231 nm); 99.05 area-% (sum of all peaks, HPLC 323 nm)
- Content: 97.5 g/100 g (100 g/100 g minus water content)
- Identity: Confirmed
- Homogeneity: The test substance was homogeneous by visual inspection.
- Storage stability: Guaranteed by the sponsor.

ADDITIONAL TEST-SUBSTANCE INFORMATION
- Physical state / color: Solid / black
- Storage conditions: Room temperature
Species:
mouse
Strain:
CBA
Remarks:
CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.1 g – 23.1 g
- Housing: Polycarbonate cages type MII with mesh wire tops, supplied by BECKER & Co., Castrop-Rauxel, Germany
- No. of animals per cage: 1 animal
- Diet: Kliba mouse/rat maintenance diet “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Drinking water, ad libitum
- Bedding: Dust-free wooden bedding was used in this study.
- Acclimation period: at least 5 days before the first test-substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
AOO was used as the vehicle because good homogeneity of the preparation was achieved.
Concentration:
10%, 25% and 50% in vehicle
No. of animals per dose:
5
Details on study design:
PRETEST
To determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP but without a GLP status) was performed.

CONDUCT OF THE STUDY
The study comprised three treatment groups and a vehicle control group. Each group consisted of 5 mice.
- Randomization: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.:Combinatorial Algorithms, Academic Press, New York,San Francisco, London, 1978, pp. 62 – 64“.
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 µL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 μCi of 3H-thymidine1 in 250 μL of sterile saline.
- ³H-thymidine incorporation into cells suspensions: After the terminal procedure the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) in an ice-water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 6 mL of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter. The remaining cell suspensions were washed twice with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a β-scintillation counter.

EVALUATION OF RESULTS
In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test substance to the skin of the dorsal surface of both ears is determined by measuring 3H-thymidine incorporation into the lymph node cells. Additional parameters used to characterize the response are, lymph node cell count and, to a certain extent, lymph node weight. Because irritation by the test substance may also induce lymph node responses, the weights of ear punches taken from the area of test-substance application are determined as an indicator for inflammatory ear swelling due to any irritant action of the test substance.
A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.
Positive control substance(s):
other: Alpha-Hexylcinnamaldehyde
Statistics:
Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.

Further statistical analysis:
Statistical test: WILCOXON - Test
Parameter: 3H-thymidine incorporation, cell count, lymph node weight and ear weight
Parameter:
SI
Value:
1
Test group / Remarks:
vehicle
Parameter:
SI
Value:
0.76
Test group / Remarks:
concentration 10%
Parameter:
SI
Value:
0.64
Test group / Remarks:
concentration 25%
Key result
Parameter:
SI
Value:
0.81
Test group / Remarks:
concentration 50%
Cellular proliferation data / Observations:
Results from main test:
- There was no increase in lymph node weights at all concentrations.
- All test-substance preparations did not cause any excessive increase in ear weights (> 25%), demonstrating the absence of ear skin irritation.
- The expected body weight gain was generally observed in the course of the study.
- Local findings: Black discolored feces was observed at the 10% concentration on study day 5 and at 25% and 50% on study days 2 and 5. Slight black discolored ear skin was noted in all animals treated with the 50% concentration.
- No signs of systemic toxicity were noticed in all animals during general observation

Detailed data on individual animals:

Test group

Treatment

Animal No

Mean value of ³H-thymidine incorporation [DPM/Lymph Node Pair]

1

Vehicle AOO

1

404.6

2

1156.4

3

934.5

4

3035.8

5

889.6

2

10% in AOO

6

996.9

7

1663.3

8

669.4

9

952.3

10

583.1

3

25% in AOO

11

635.3

12

579.2

13

642.5

14

1357.1

15

917.6

4

50% in AOO

16

1444.0

17

1425.0

18

878.5

19

730.3

20

705.8

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation in vivo (substance itself, EC 916-865-0)

The skin sensitising potential of the test substance was assessed using the LLNA according to the OECD guideline 429. Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% w/w preparations of the test item in AOO 4+1 v% or with the vehicle alone. The study used 3 test groups and 1 control group. Each test animal was treated with 25 μL per ear of the appropriate test-item preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. Black discolored feces was observed at the 10% concentration on study day 5 and at 25% and 50% on study days 2 and 5. Slight black discolored ear skin was noted in all animals treated with the 50% concentration. No signs of systemic toxicity were noticed in any of the animals during general observation. When applied as 10%, 25% and 50% preparations in AOO, the test item did not induce a biologically relevant or statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, there were no biologically relevant or statistically significant increases in lymph node cell counts at all concentrations. Further, no increase in lymph node weights at all concentrations was observed. All test-item preparations did not cause any excessive increase in ear weights (>25%), demonstrating the absence of ear skin irritation. The increase at 50% was statistically significant. Thus it is concluded that the test item does not exhibit a skin sensitizing potential in the LLNA under the selected test conditions (BASF, 2017).

Additional information

Sensitisation data (human): Insult patch test (Orasol Black CN, CAS 12237-23-9)

Additional information is available based on a repeated insult patch test assessing a constituent of the substance (Orasol Black CN) of the test item on 200 volunteers. Volunteers are selected as general type of population. The patch test was performed in a series of 8 48-hour applications. The structural analogue was applied under occlusion for effective contact periods of 2 -days duration using patches of 3x3 cm. If no reactions occurred, the structural analogue was re-applied immediately for another 48 -hour period. The following scoring system was used: 0 = No reactions; 1+ = Slight erythema; 2+ = Marked erythema; 3+ = Marked erythema, oedema, with or without a few vesicles; 4+ = Marked erythema, edema, with vesicles and oozing. Based on the available information, no visible skin changes signifying reaction to injury were observed in any of the 200 subjects. Since this is a structural analogue of the target substance (EC 916 -865 -0), the target substance is also not expected to cause visible skin changes to humans (Ciba-Geiga Ltd, 1974).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information, classification for skin sensitisation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.