Fatty acids, montan-wax, stearyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 30 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Jul 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

not specified

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:

Fatty acids, montan-wax, stearyl esters

EC Number:

269-642-9

EC Name:

Fatty acids, montan-wax, stearyl esters

Cas Number:

68308-30-5

Molecular formula:

C18 H38 O - C66 H130 O4

IUPAC Name:

Fatty acids C22-30 (even numbered), octadecyl alcohol esters

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 20.9 - 21.9 g
- Housing: Animals were housed in groups in Type II polypropylene / polycarbonate cages with bedding.
- Diet: Ssniff SM R/M-15mm "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 523 7816, produced by ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.8 - 24.2
- Humidity (%): 30 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 24 Oct 2012 To: 30 Oct 2012

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

(DMF)

Concentration:

10, 25 and 50% (w/v)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: 2 female mice were treated by daily application of 25 μL of the test substance in concentrations of 25 and 50% on the dorsal surface of the ear, for 3 consecutive days. The body weight was recorded on Day 1 prior to dosing and on Day 6.
- Compound solubility: Based on the observation of the solubility test, the maximum available concentration was 50% (w/v).
- Irritation: The animals were observed for local skin irritation to the application site once a day. Erythema were not observed.
- Systemic toxicity: The animals were observed for signs of toxicity once a day. No clinical signs of toxicity or mortality were noted.
- Ear thickness measurements: Ear thickness was measured using a thickness gauge on Day 1, 3 and 6. At 25 and 50%, no increase of the ear thickness was determined on Day 1, 3 and 6, respectively.
- Erythema scores: Draize scoring system

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation counting
- Criteria used to consider a positive response: Exposure to at least one concentration of the test substance resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI). 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.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test substance was applied to the entire dorsal surface of each ear of each mouse on Day 1, 2 and 3 in concentrations of 10, 25 and 50% in DMF. On Day 6 an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each mouse. Five hours later, the draining auricular lymph node of each ear was excised into PBS and pooled per experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe gauze and rinsed with PBS. To precipitate out the radioactive material, the pellet was resuspended in 5% trichloroacetic acid. The precipitates were incubated for approximately 18 h at 2 - 8 °C, centrifuged, resuspended in 1 mL TCA and transferred to 10 mL scintillation fluid before β-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control substance (25% hexyl cinnamic aldehyde in DMF) was considered to be a sensitiser under the conditions of the test (SI 12.9).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

50% (w/v)

Results 2

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

25% (w/v)

Results 3

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

10% (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 10, 25 and 50% treatment group was 0.8, 1.1 and 1.8%, respectively.

EC3 CALCULATION
The EC3 value could not be calculated, since all SI values are below the threshold value of 3.

CLINICAL OBSERVATIONS
No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

BODY WEIGHTS
No treatment related effects were observed on animal body weights.

Any other information on results incl. tables

Table 1: Summary of results

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 (w/v) % TCA)	32.5	-	-	-	-
Negative (vehicle) control (DMF)	1478	1445.5	8	180.7	1.0
50 (w/v) % in DMF	2653	2620.5	8	327.6	1.8
25 (w/v) % in DMF	1646	1613.5	8	201.7	1.1
10 (w/v) % in DMF	1222	1189.5	8	148.7	0.8
Positive control (25 (w/v) % HCA in DMF)	18716	18683.5	8	2335.4	12.9

TCA: Trichloroacetic acid

HCA: Hexyl cinnamic acid

DPM: Disintegrations per minute

DPN: Disintegrations per node

Applicant's summary and conclusion

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified