Acetic acid, 2-(ethylamino)-2-oxo-, (1R,2S,5R)-5-methyl-2-(1-methylethyl)cyclohexyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-16 to 2009-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study. Deviations according to OECD 429 (2010) with no effect on the results: - pre-test: no body weight and ear thickness was measured - historical positive control protocol missing - SD was not given for the dpm

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008-05-31

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2010-07-22

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2008-11-12

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (ethylcarbamoyl)formate

EC Number:

700-185-4

Cas Number:

1122460-01-8

Molecular formula:

C14H25NO3

IUPAC Name:

(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (ethylcarbamoyl)formate

Test material form:

liquid: viscous

Details on test material:

- Molecular formula: C14H25NO3
- Molecular weight: 255.36g/mol
- Physical state: liquid
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland, P.O. Box 6174, 5960 AD Horst / Netherlands
- Age at study initiation: 8 - 12 weeks at the beginning of treatment
- Weight at study initiation: 18.1 – 21.8 g at the beginning of treatment
- Housing: group housing in Makrolon Type-II cages with standard softwood bedding.
- Diet (ad libitum): pelleted standard Kliba 3433 mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland)
- Water (ad libitum): community tap water from Itingen/Switzerland
- Acclimation period: 7 days prior to the start

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30- 70%
- Air changes: 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (at least 8 hours music during the light period)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10, 25 and 50 % concentration of the test item

No. of animals per dose:

4 female mice

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50 % and 25 % (w/v) each in acetone/olive oil (4:1 v/v) on three consecutive days. In the pre-test clinical signs were recorded within 4 ± 2 hours and 24 ± 4 hours after each application as well as on the day corresponding to the day of preparation of the main experiment. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 10 %, 25 % and 50 % (w/v) in acetone/olive oil (4:1 v/v). The top dose is the highest technically achievable concentration while avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
Each test group of mice was treated by topical application to the dorsal surface of each ear lobe with the test item at the different concentrations in the vehicle. The application volume of 25 μL was spread over the entire dorsal surface of each ear lobe (Ø about 8 mm) once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle.
Five days after the first topical application, all mice were administered with 250 μL of phosphate-buffered saline (PBS) containing 78.70 μCi/mL 3HTdR (equal to 19.68 μCi 3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by inhalation of CO2.
The draining lymph nodes were rapidly excised and pooled in PBS for each experimental group (8 lymph nodes per group). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a gauze (100-200 μm mesh size). After washing twice with approximately 10 mL phosphate buffered saline the lymph node cells were resuspended in approximately 3 mL 5% trichloroacetic acid and incubated at approximately + 5 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 1 mL 5% trichloroacetic acid and transferred to glass scintillation vials with 10 mL of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5% trichloroacetic acid.

INTERPRETATION OF RAW DATA:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of the test groups relative to that recorded for the control group (Stimulation Index S.I.). Before dpm/lymph node values were determined, the mean scintillation-background dpm was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: once daily from the start of acclimatisation to the termination of the in-life phase.
Body weights: at the start of acclimatisation, on test day 1 (prior to the 1st application) and on test day 6 (prior to 3HTdR injection).
Clinical signs (local / systemic): on the day of animal delivery and once daily from test day 1 (after the 1st application) to the termination of in-life phase.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated.

Results and discussion

Positive control results:

The proliferative capacity of the pooled lymph node cells was determined by measuring the incorporation of 3HTdR using a β-scintillation counter.
5 % concentration of the test item in acetone/olive oil (4:1 v/v): S.I. = 1.0
10 % concentration of the test item in acetone/olive oil (4:1 v/v): S.I. = 2.3
25 % concentration of the test item in acetone/olive oil (4:1 v/v): S.I. = 8.7
A dose-response relationship was observed.
A calculation of the EC3 value was performed resulting in an EC3 value of 11.6 %.
No deaths occurred during the study period.
Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.
The body weights of the animals, recorded at the start of acclimatisation, prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.
Alpha-hexylcinnamaldehyde shows an allergic potential when tested up to the concentration of 25 % (w/v) in acetone/olive oil (4:1 v/v).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The proliferative capacity of the pooled lymph node cells was determined by measuring the incorporation of 3HTdR using a β-scintillation counter. 10 % concentration: S.I. = 1.6 25 % concentration: S.I. = 1.7 50 % concentration: S.I. = 1.3 No dose-response relationship was observed. A calculation of the EC3 value was not performed because no test concentration produced a S.I. of 3 or higher.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Results based on pooled approach: - Vehicle: 1045 (dpm - background dpm); 131 (dpm/node) - 10 % v/v concentration: 1638 (dpm - background dpm ); 205 (dpm/node) - 25 % v/v concentration: 1762 (dpm - background dpm ); 220 (dpm/node) - 50 % v/v concentration: 1314 (dpm - background dpm); 164 (dpm/node)

Any other information on results incl. tables

OBSERVATION:

- Viability / Mortality: no deaths occurred during the study period.

- Clinical signs: neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

- Body weights: the body weights of the animals, recorded at the start of acclimatisation, prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this study S.I. of 1.6, 1.7 and 1.3 were determined with the test item dissolved in acetone/olive oil (4:1 v/v) at concentrations of 10%, 25% and 50% (w/v), respectively.
The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.
According to 67/548/EC and subsequent regulations, the test substance is not classified as skin sensitiser.
According to the EC Regulation No. 1272/2008 and subsequent regulations, the test substance is not classified as skin sensitiser.