dodecane-12-lactam, manufacturing of, by-products from, distillation residues

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2011 - 03 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

dodecane-12-lactam, manufacturing of, by-products from, distillation residues

EC Number:

923-400-5

IUPAC Name:

dodecane-12-lactam, manufacturing of, by-products from, distillation residues

Test material form:

solid

Details on test material:

- Name of test material: Dodecane-12-Lactam Manufacturing of, by-products from, distillation residues
- Physical state: brown solid
- Lot/batch No.: AVRIL 2011
- Expiry date: 30 April 2012
- Composition of test material: UVCB
- Storage condition: 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: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the beginning of the treatment period, the animals of the preliminary test were approximately 10 weeks old and the animals of the main test were approximately 8 weeks old
- Mean body weight at study initiation: mean body weight of 20.6 g (range: 19.6 g to 21.7 g)
- Fasting period before study: no
- Housing: the animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: the animals were acclimated to the study conditions 12 days (preliminary test) or 13 days (main test) before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h (7:00 - 19:00)

IN-LIFE DATES: 19 October 2011 to 31 October 2011.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25%

No. of animals per dose:

4 females per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility the vehicle used in the study was selected from the results of solubility assays performed by the CIT prior to the preliminary assay.
In the absence of recommendation from the Sponsor, in order to select the most appropriate vehicle, the solubility assay first started at the concentration of 50%, according to the OECD Guideline. None of the vehicle from the selection of vehicles provided a homogeneous suspension at 50%. A homogeneous suspension was obtained with at the concentration of 25% in Acetone/Olive Oil (4/1, v/v) (AOO).

- Irritation: none.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: T
The test item was administered as a suspension in the vehicle.
The test item was mixed under magnetic agitation with the required quantity of vehicle. The test item concentrations were expressed in % (w/v).
The positive control was prepared at the concentration of 25% (v/v) in AOO.
Dosage forms preparations were prepared by the CIT Pharmacy extemporaneously on the day of each administration.
The dosage forms were stored at room temperature and delivered to the study room in brown flasks.

The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

Positive control substance(s):

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

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Test item at the concentration of 1%, stimulation index: 1.87 Test item at the concentration of 2.5%, stimulation index: 1.99 Test item at the concentration of 5%, stimulation index: 2.15 Test item at the concentration of 10%, stimulation index: 2.55 Test item at the concentration of 25%, stimulation index: 1.23

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per group: Group 1: Vehicle: 771 Group 2: 1%: 1442 Group 3: 2.5%: 1534 Group 4: 5%: 1654 Group 5: 10%: 1968 Group 6: 25%: 950

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

Executive summary:

The objective of this GLP study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA) according to OECD 429. Evaluation of local irritation was also carried out in parallel.

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, 28 female CBA/J mice were allocated to 7 groups / five treated groups of four animals receiving the test item at the concentration of 1, 2.5, 5, 10 or 25% in Acetone/Olive Oil (AOO) (vehicle),  one negative control group of four animals receiving the vehicle,  one positive control group of four animals receiving the positive control item,a-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in a mixture acetone/olive oil (4/1; v/v).

During the induction phase, the test item, vehicle or positive control item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6. No unscheduled deaths occurred and no clinical signs were observed during the preliminary or main test. No cutaneous reactions and no increase in ear thickness were observed at any of the tested concentrations.

 The stimulation index was range between 2.23 and 2.55, therefore the test item did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.