Registration Dossier

Administrative data

Description of key information

OECD 429 (LLNA, BASF 2018): positive, EC 3 = 36.5%

OECD 442C, 442D, 442E (in vitro Skin Sens Battery, BASF 2017): positve

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:
weight of evidence
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE; Experimental Toxicology and Ecology; Ludwigshafen; Germany
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Purity: 98.1 area-% (GC, DB-1 capillary); 99.1 area-% (GC, DB-Wax UI capillary)
Homogeneity: The test substance was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Physical state / color: Liquid / colorless, clear
Storage conditions: Room temperature, avoid temperatures > 60°C
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, The Netherlands
- Age at study initiation: 8 weeks (main test)
- Weight at study initiation: 17.1 – 21.2 g (main test)
- Housing: 1 animal/cage
- Diet: ad libitum; Kliba mouse/rat maintenance diet “GLP" by Provimi Kliba SA,Switzerland.
- Water: ad libitum
- Acclimation period: min. 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
methyl ethyl ketone
Concentration:
25% test substance in MEK
50% test substance in MEK
undiluted test substance
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Yes
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Lymph node weight: Yes

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
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“.

TREATMENT PREPARATION AND ADMINISTRATION:
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 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
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-thymidine in 250 µL of sterile saline.

Control group 0 untreated
Control group 1 treated with the vehicle
Group 2 25% test substance in vehicle
Group 3 50% test substance in vehicle
Group 4 undiluted test substance

- Parameters assessed:
Determination of ear weight:
Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal.

Removal and weight determination of the lymph nodes:
Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.

Preparation of cell suspension and determination of cell count:
After weight determination, the pooled lymph nodes of each animal were stored in phosphate buffered saline (PBS) and a single cell suspension was prepared by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into phosphate-buffered physiological saline. The cell count was determined using a Casy®-Counter.

Measurement of 3H-thymidine incorporation of the lymph node cells:
The remaining cell suspensions were washed with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was measured in a β-scintillation counter.

- Criteria used to consider a positive response:
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 are 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
3H-thymidine incorporation, cell count, lymph node weight and ear weight: Wilcoxon - Test
Key result
Parameter:
EC3
Value:
36.5
Test group / Remarks:
3H-thymidine incorporation
Cellular proliferation data / Observations:
BODY WEIGHTS
No relevant influence on the mean body weights was observed during the study.

CLINICAL SIGNS
No signs of systemic toxicity were noticed in all animals during general observation.

LOCAL FINDINGS
No local findings were observed during the observation period.

As the concurrent vehicle control value was lower than control values usually measured with the same vehicle, vehicle-related historical control data collected over an appropriate period were used for the evaluation of the results in addition to the concurrent control values.

Test Group  Treatment ³H-thymidine
incorporation
Stimulation Index
Cell Count
Stimulation Index
Lymph Node Weight
Stimulation Index
Ear Weight
Stimulation Index
    vs.
concurrent
vehicle/
untreated
control
vs.
historical
vehicle/
untreated
control
vs.
concurrent
vehicle/
untreated
control
vs.
historical
vehicle/
untreated
control
vs.
concurrent
vehicle/
untreated
control
vs.
historical
vehicle/
untreated
control
vs.
concurrent
vehicle/
untreated
control
vs.
historical
vehicle/
untreated
control
0

untreated1

1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
1 vehicle MEK2 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
2 25% in MEK2 4.57 ## 1.82 1.75 # 1.56 1.34 ## 1.45 1.02 1.03
3 50% in MEK2 11.02 ## 4.38 2.45 ## 2.18 1.59 ## 1.72 1.03 1.04
4

undiluted test substance1

15.61 ##

7.75

2.84 ##

2.28

2.03 ##

2.17

1.06

1.14

1 Stimulation index calculated as test group x / test group 0 (concurrent or historical untreated control)

2 Stimulation index calculated as test group x / control group 1 (concurrent or historical vehicle control)

The statistical evaluations (for comparision to concurrent vehicle and untreated groups, only) were performed using the WILCOXON-test ( # for p ≤ 0.05, ## for p ≤ 0.01 )

Executive summary:

The skin sensitizing potential of pyranyl acetate pure was assessed by using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 25% or 50% (w/w) preparations of the test substance in methyl ethyl ketone (MEK), with the undiluted test substance, with the vehicle alone or remained untreated. The study used 3 test groups and 2 control groups. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or of the undiluted test substance applied to the dorsal surfaces of both ears on three consecutive days. One control group was treated with 25 μL per ear of the vehicle alone and one control group remained untreated. As the concurrent vehicle control value was lower than control values usually measured with the same vehicle, vehicle-related historical control data collected over an appropriate period were used for the evaluation of the results in addition to the concurrent control values.

No signs of systemic toxicity were noticed in all animals during general observation.

When applied 25% and 50% preparation in MEK as well as when applied undiluted the test substance induced a biologically relevant (increase above the cut-off Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The 25% and 50% test-substance preparation and the undiluted test substance induced a biologically relevant and statistically significant response (increase to 1.5-fold or above ofcontrol value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant increases in lymph node weights were noted after application of the 25% and 50% concentration and of the undiluted test substance. The test-substance concentrations did not cause increases (SI1.25) in ear weights demonstrating the absence of relevant ear skin irritation.Thus, it is concluded that pyranyl acetate pure exhibits a skin sensitizing potential in theMurine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >25% <50%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count were calculated by linear regression from the results of these concentrations to be 36.5% and 22.7%, respectively.

 

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

In vitro studies:

In a Direct peptide reactivity assay (DPRA) according to OECD TG 442C, pyranyl acetate pure was dissolved in acetonitrile and mixed with a cysteine- and a lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by pyranyl acetate pure was determined by HPLC-UV.

The samples of the test substance with cysteine-peptide were solutions at the time of preparation and after 24 hours. The samples of the test substance with the lysine-peptide were emulsions at the time of preparation and after 24 hours.The mean C-peptide depletion, caused by the test substance was determined to be 2.68%. The mean K-peptide depletion, caused by the test substance was determined to be -4.21%. Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

 

The keratinocyte activating potential of test substance pyranyl acetate pure was evaluated in the LuSens assay according to OECD TG 442D. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours. The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 137μg/mL (experiment 4) and 135μg/mL (experiment 5), respectively. In summary, after 48 hours of exposure to test substance pyranyl acetate pure luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance pyranyl acetate pure has a keratinocyte activating potential.

 

In a h-CLAT according to OECD TG 442E, pyranyl acetate pure was dissolved in DMSO and tested at 285-1021 µg/mL in two valid experiments. After 24 hourincubation with human monocytic leukemia cell line THP-1, membrane marker expression (CD86 / CD54) was measured by flow cytometry. Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable. The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 358μg/mL (experiment 1). Calculation of an EC200% for experiment 2 was not applicable as fold inductions above 200% were obtained in all tested concentrations. After 24 hours of exposure to test substance pyranyl acetate pure CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance pyranyl acetate pure induces dendritic cell activation.

 

Addressing key events of the adverse outcome pathway (AOP) for skin sensitizationpyranyl acetate pureis predicted to be a skin sensitizerin the in vitro Skin Sensitization Turnkey Testing Strategy.

 

 

In vivo studies:

The skin sensitizing potential of pyranyl acetate pure was assessed by using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 25% or 50% (w/w) preparations of the test substance in methyl ethyl ketone (MEK), with the undiluted test substance, with the vehicle alone or remained untreated. The study used 3 test groups and 2 control groups. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation or of the undiluted test substance applied to the dorsal surfaces of both ears on three consecutive days. One control group was treated with 25 μL per ear of the vehicle alone and one control group remained untreated. As the concurrent vehicle control value was lower than control values usually measured with the same vehicle, vehicle-related historical control data collected over an appropriate period were used for the evaluation of the results in addition to the concurrent control values.

No signs of systemic toxicity were noticed in all animals during general observation.

When applied 25% and 50% preparation in MEK as well as when applied undiluted the test substance induced a biologically relevant (increase above the cut-off Stimulation Index of 3), statistically significant and concentration-dependent increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes. The 25% and 50% test-substance preparation and the undiluted test substance induced a biologically relevant and statistically significant response (increase to 1.5-fold or above ofcontrol value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. In addition, statistically significant increases in lymph node weights were noted after application of the 25% and 50% concentration and of the undiluted test substance. The test-substance concentrations did not cause increases (SI1.25) in ear weights demonstrating the absence of relevant ear skin irritation. Thus, it is concluded that pyranyl acetate pure exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >25% <50%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count were calculated by linear regression from the results of these concentrations to be 36.5% and 22.7%, respectively.

 

 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

The present data on dermal sensitization fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a classification with "Skin sensitisation" (Category 1B) is warranted.