4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-31 March 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

GLP study conducted in compliance with OECD Guideline No. 429. However, isomers ratio is not reported.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

24 April 2002.

Deviations:

yes

Remarks:

acceptable deviation: proliferation of lymphocytes determined by cell counting instead of radioactivity

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Directive 2004/73/EC.

Deviations:

yes

Remarks:

acceptable deviation: proliferation of lymphocytes determined by cell counting instead of radioactivity

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2007-01-11 / Signed on 2007-02-21.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (2R,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2S,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Molecular formula:

C11H22O2

IUPAC Name:

Reaction mass of (2R,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2S,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Constituent 2

Reference substance name:

Reaction mass of (2S,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2R,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Molecular formula:

C11H22O2

IUPAC Name:

Reaction mass of (2S,4R)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol & (2R,4S)-4-methyl-2-(pentan-3-yl)tetrahydro-2H-pyran-4-ol

Test material form:

liquid

Details on test material:

- Physical state: Colourless to pale yellow, limpid/translucent liquid

Specific details on test material used for the study:

- Test item was considered at 100% for the study.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J@Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle).
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 20-23 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food, ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 41-60 %
- Air changes: Approximately fifteen changes per hour
- Photoperiod: 12 h dark / 12 h light

- IN-LIFE DATES: 19-31 March 2008

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Preliminary screening test: 100%
Main test: 100%, 50% or 25% (v/v) in Acetone/Olive oil (4:1)

No. of animals per dose:

Preliminary screening test: One animal/dose
Main test: 4 animals/dose

Details on study design:

PRELIMINARY STUDY
- Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at 100%, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed once daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
- Irritation: No signs of systemic toxicity were noted. No cutaneous reaction was observed. Based on this information, the dose levels selected for the main test were 25% and 50% v/v in Acetone/Olive oil (4:1) and 100%.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. Other relevant criteria such as dose-response and irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI < 1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of control or test substance was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. A single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS (Ca2+ / Mg2+ - free) containing 0.5% BSA into a well of a multi-well 6. 10 μL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counting using a cell counter (Beckman Coulter Z2). For the run, the lower size selected was 5 μm and the upper size selected was 15 μm (the average size of a lymphocyte is 8 μm).
The proliferation response of lymph node cells was expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

None

Results and discussion

Positive control results:

Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α- Hexylcinnamaldehyde, as a solution in dimethylformamide at concentrations of 5%, 10% and 25% (v/v). With EC1.4 = 4.86, α-Hexylcinnamaldehyde is considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.94

Test group / Remarks:

25% in Acetone/Olive oil (4:1)

Results 2

Key result

Parameter:

SI

Value:

1.19

Test group / Remarks:

50% in Acetone/Olive oil (4:1)

Results 3

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

100% in Acetone/Olive oil (4:1)

Cellular proliferation data / Observations:

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
- No stimulation index of more than 1.4 was recorded whatever the tested concentrations (25% (w/w), 50% (w/w) in Acetone/olive oil (4:1) and 100%).
- The Stimulation Index (SI) calculated by pooled approach was respectively 0.94, 1.19 and 1.03 for the treated group at 25%, 50% and 100%.

LOCAL IRRITATION
- No cutaneous reaction was observed.
- No significant increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%. Therefore, the test item must be considered “non-irritant” at the three concentrations.

CLINICAL OBSERVATIONS:
- No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test.

BODY WEIGHTS
- Bodyweight changes of the test animals between day 1 and day 6 were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, the test substance is not classified as sensitiser according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, three groups of CBA/J (CBA/J@Rj) strain mice (4 females/dose) were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the undiluted test item and the test item as a solution in Acetone/Olive oil (4:1) at concentrations of 25% and 50% (v/v). A further group of four animals was treated with Acetone/Olive oil (4:1). At D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The test concentrations for the main study were determined from a preliminary study tested at 100 %.Based on the results of preliminary study, the dose levels selected for the main test were 25% and 50% v/v in Acetone/Olive oil (4:1) and 100%.

 

No mortality and no signs of systemic toxicity were noted in the test and controls animals during the test. No cutaneous reaction was observed. No significant increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%. Therefore, the test item must be considered “non-irritant” at the three concentrations. No stimulation index of more than 1.4 was recorded whatever the tested concentrations (25% (w/w), 50% (w/w) in Acetone/olive oil (4:1) and 100%). The Stimulation Index (SI) calculated by pooled approach was respectively 0.94, 1.19 and 1.03 for the treated group at 25%, 50% and 100%.

 

Under the test conditions, the test substance is not classified as sensitiser according to the criteria of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin sensitisation endpoint.