2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE; Experimental Toxicology and Ecology; Ludwigshafen; Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate

EC Number:

942-380-9

Cas Number:

131796-64-0

Molecular formula:

C12H22O3

IUPAC Name:

2-isobutyl-4-methyltetrahydro-2H-pyran-4-yl acetate

Test material form:

liquid

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

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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

Results and discussion

In vivo (LLNA)

Results

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.

Any other information on results incl. tables

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	untreated^1	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
1	vehicle MEK^2	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1.00
2	25% in MEK^2	4.57 ##	1.82	1.75 #	1.56	1.34 ##	1.45	1.02	1.03
3	50% in MEK^2	11.02 ##	4.38	2.45 ##	2.18	1.59 ##	1.72	1.03	1.04
4	undiluted test substance^1	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 )

Applicant's summary and conclusion

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 (SI≥1.25) 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.