Fatty acids, C16-18 (even numbered), ammonium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 26, 2012 - October 25, 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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:

diammonium hexadecanoate octadecanoate

EC Number:

939-066-9

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).

IUPAC Name:

diammonium hexadecanoate octadecanoate

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: 8 – 9 weeks
- Weight at study initiation: 17-23 g
- Housing: Groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

100, 25 and 12.5% (diluted with DMSO, v/v)

No. of animals per dose:

5 mice per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item in the vehicle was found to be 25% in DMSO.
- Irritation: Four animals were treated by topical application with the test item on three consecutive days at 100, 25 and 0% test item in DMSO. Neither signs of systemic toxicity nor signs of irritation at the application site could be detected in any animal.
- Lymph node proliferation response: No assessed on the prescreen test. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance is regarded as a 'sensitizer' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Five days after the first application, 3H-methyl thymidine by intravenous injection was administered. 5 hours later, mice were sacrificed, the draining auricular lymph nodes excised and the pooled cell suspension prepared.
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Positive control substance(s):

other: P-Phenylenediamine (CAS 106-50-3) - Historical data

Results and discussion

Positive control results:

The laboratory conducts LLNA assay regularly and has an established historical positive-control database based on the substance p-Phenylenediamine for which reproducible and accureate positive results were obtained, and therefore, the substance is suitable as a positive control.
The recent reliability check was performed in October 2012. The raw data of this study are kept in the BSL archives. Stimulation index for 1% positive control on three consecutive days was 9.6 ± 3.3.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

(12.5% conc.)

Value:

0.9

Remarks on result:

other: The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Results 2

Key result

Parameter:

SI

Remarks:

(25% conc.)

Value:

0.7

Remarks on result:

other: The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Results 3

Key result

Parameter:

SI

Remarks:

(100% conc.)

Value:

0.6

Remarks on result:

other: The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

Any other information on results incl. tables

Radioactive Determination of the Test Substance Groups:

	CPM	TEST ITEM	CONC. [%]	DPM	DPM mean back ground	DPM/NODE	SI
MV	1332.6	Negative control DMSO		2730.8	2708.2	1354.1	1.0
SD	445.1			911.1	911.1	455.5
MV	1332.6	Test ítem in DMSO	12.5	2553.0	2530.4	1265.5	0.9
SD	445.1			140.0	140.0	70.0	0.1
MV	1332.6	Test ítem in DMSO	25.0	1991.6	1969.0	984.5	0.7
SD	445.1			408.7	408.7	204.3	0.2
MV	1332.6	Test ítem	100	1684.8	1662.2	831.1	0.6
SD	445.1			311.3	311.1	155.7	0.1
MV	1332.6	Background Szinti and TCA		22.6	0.0	0.0	0.0
SD	445.1			1.9

CPM = counts per minute; Conc. = concentration; DPM = disintegrations per minute; SD = standard deviation; MV = mean value; Szinti = scintillation fluid; TCA = trichloroacetic acid

All animals survived throughout the test period without showing any clinical signs.

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

Applicant's summary and conclusion

Interpretation of results:

other: No category (CLP Regulation EC no. 1272/2008)

Conclusions:

Fatty acids, C16-18 (even numbered), ammonium salts was determined to be not skin sensitising under test conditions in the LLNA assay since none of the three tested concentrations reached the stimulation index of 3.

Executive summary:

A Local Lymph Node Assay (LLNA) was performed on test item Fatty acids, C16 -18 (even numbered), ammonium salt in accordance with OECD Guideline 429. Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 12.5% (v/v), 25% (v/v) each diluted with DMSO (v/v) and 100% (undiluted test item). 5 female CBA mice per dose were treated by topical application of 25 μL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The 3H-methyl thymidine - incorporation into lymph node cells of the test group animals relative to the control goup animals were measure (Stimulation index). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed. None of the three tested concentrations of the test item reached the stimulation index of 3 and the EC3 value (derived by linear interpolation) could not be calculated. The test substance Fatty acids, C16 -18 (even numbered), ammonium salt was determined to be not skin sensitising.