Sebacic acid, compound with hexane-1,6-diamine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 August 2012 to 08 January 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 429), well performed without any deviation.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

Remarks:

(Date: 19 DEC 2012)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sebacic acid, compound with hexane-1,6-diamine (1:1)

EC Number:

229-177-4

EC Name:

Sebacic acid, compound with hexane-1,6-diamine (1:1)

Cas Number:

6422-99-7

Molecular formula:

C10H18O4.C6H16N2

IUPAC Name:

decanedioic acid; hexane-1,6-diamine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): sebacic acid, compound with hexane-1,6-diamine (1:1)
- Physical state: white powder
- Stability under test conditions: the stability of the test item is the responsability of the sponsor.
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: eight to twelve weeks old.
- Weight at study initiation: the weight range of 15 to 23 g
- Housing: individually
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK.
- Water (e.g. ad libitum): tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Study design: in vivo (LLNA)

Vehicle:

other: Pluronic L92

Concentration:

- Preliminary test: 25% w/w in 1% pluronic L92 in distilled water.
- Main test: 25%, 10% and 1% w/w in 1% pluronic L92 in distilled water.

No. of animals per dose:

1 female per dose for the preliminary test.
4 females per dose for the main test.

Details on study design:

RANGE FINDING TESTS:

- Compound solubility: the test item was prepared as a solution in 1% pluronic L92 in distilled water (BASF, Batch: WPAG522B). This vehicle was chosen as all other vehicles were unsuitable for dosing which was at a concentration of 25% w/w.

- Irritation: Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse at a concentration of 25% w/w in 1% pluronic L92 in distilled water. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the dose levels selected for
the main test were 25%, 10% and 1% w/w in 1% pluronic L92 in distilled water.

- Lymph node proliferation response: not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- 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 in a threefold or greater increase in 3HTdR incorporation compared to control values. Together with consideration of a dose-response relationship other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.
Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
The animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR. Once injected, the mice were left for 5 hours.

Positive control substance(s):

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

Results and discussion

Positive control results:

The stimulation Index for the positive control tested on June 2012 at a concentration of 25% v/v in 1% pluronic L92 in distilled water was 7.20. This gave a positive result for sensitisation potential.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: No notable lymphoprofileration was noted with the test item at any tested concentrations. The observed stimulation index values were 1.75, 1.30 and 1.84 at concentrations of 25, 10 and 1 ( % w/w) in 1% pluronic L92 in distilled water respectively.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See Table 7.4.1/1

Any other information on results incl. tables

Table 7.4.1/1 Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%w/w) in 1% pluronic L92 indistilled water	Dpm/group	dpm/Node^a	Stimulation Index^b	Result
Vehicle	9850.00	1231.25	na	na
1	18124.54	2265.57	1.84	negative
10	12780.17	1597.52	1.30	negative
25	17237.32	2154.67	1.75	negative

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =   Not applicable

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the experimental conditions of this study, the test item, sebacic acid, compound with hexane-1,6-diamine (1:1), gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Executive summary:

In a dermal sensitization study performed according to the OECD test guideline No. 429 and in compliance with Good Laboratory Practice (Henzell, 2012), sebacic acid, compound with hexane-1,6-diamine (1:1) was tested in female CBA/Ca strain mice using the Local Lymph Node Assay.

A vehicle determination was performed with the test item before preliminary testing commenced. All vehicles were unsuitable in the Local Lymph Node Assay apart from 1% pluronic L92 in distilled water which showed suitability at a concentration was 25% w/w.

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

In the main test, three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test item as a solution in 1% pluronic L92 in distilled water at concentrations of 25%, 10% or 1% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone. The test item solutions were applied on the dorsal surface of ears of experimental animals (25µl/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine ((3H-TdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or systemic clinical signs were observed during the study. No treatment related effects were observed on animal body weights in any treated groups. No notable lymphoprofileration was noted with the test item at any tested concentrations. The observed Stimulation Index values were 1.75, 1.30 and 1.84 at concentrations of 25, 10 and 1 (% w/w) in 1% pluronic L92 in distilled water respectively.

The Stimulation Index for the positive control (α‑Hexylcinnamaldehyde, tech., 85%, tested on June 2012) at a concentration of 25% v/v in 1% pluronic L92 in distilled water was 7.20. This gave a positive result for sensitisation potential.

Under the experimental conditions of this study, the test item, sebacic acid, compound with hexane-1,6-diamine (1:1), gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties. Based on these results, no classification is required according to the Regulation (EC) No. 1272/2008 (CLP) and the Directive 67/548/EEC criteria.