Bis(2-ethylhexyl) azelate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 - 25 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study (Draft report)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, London, UK

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) azelate

EC Number:

203-091-7

EC Name:

Bis(2-ethylhexyl) azelate

Cas Number:

103-24-2

Molecular formula:

C25H48O4

IUPAC Name:

bis(2-ethylhexyl) azelate

Details on test material:

- Name of test material (as cited in study report): Diethylhexyl Azelate
- Physical state: pale yellow liquid
- Analytical purity: no data
- Lot/batch No.: 558638
- Expiration date of the lot/batch: 2014-06-20
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: 2014C Teklad Global Rodent (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Preliminary screening test: 100%
Main assay: 25% (v/v), 50% (v/v) and 100%

No. of animals per dose:

Preliminary screening test: 1
Main assay: 4

Details on study design:

RANGE FINDING TESTS: In a preliminary screening test, one mouse was treated by daily application of 25 µL of the undiluted test substance (100% concentration) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6 and clinical signs of toxicity were recorded. Local skin irritation was scored daily and thickness of each ear was measured pre-dose on Day 1, post-dose on Day 3 and on Day 6. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- Compound solubility: The compound was well soluble in acetone/olive oil (4:1 v/v) at the required concentration.
- Clinical signs: No clinical signs of toxicity were noted at a concentration of 100% (undiluted test substance).
- Irritation: No visual local skin irritation and no irritation indicated by a ≥ 25% increase in mean ear thickness were noted after treatment. Based on these results, the undiluted test substance (100%) and concentrations of 50% and 25% (v/v) in acetone/olive oil (4:1 v/v) were selected for the main assay.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: The test substance will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in ³H-methyl thymidine (³H-TdR) incorporation compared to control values. Any test item failing to produce a threefold or greater increase in ³H-TdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: The animals were treated with the undiluted test substance (100%), test substance at concentrations of 25% and 50% (v/v) in acetone/olive oil (4:1 v/v) or the vehicle alone by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1-3). Five days following the first topical application of the test substance or vehicle (Day 6), each animal was injected with 250 µl of phosphate buffered saline (PBS) containing 20 µCi ³H-TdR (concentration: 80 µCi/mL, specific activity: 2.0 Ci/mmol) via the tail vein. Five hours following administration of ³H-TdR, animals were sacrificed and draining auricular lymph nodes were excised and pooled for each experimental group (total: 8 nodes/group). A single cell suspension of pooled lymph node cells was prepared in PBS by gentle mechanical disaggregation through 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish and each lymph node cell suspension was transferred into a centrifuge tube. The petri dish was washed with PBS to remove all remaining lymph node cells, and pooled lymph node cells were pelleted by centrifugation. The pellet was resuspended in PBS and re-pelleted. To precipitate macromolecules incorporating radioactive material, the pellet was resuspended in 5% trichloroacetic acid (TCA). After approx. 18 h incubation at 4°C, the precipitates were recovered by centrifugation, resuspended in TCA and transferred to scintillation fluid. ³H-TdR incorporation was measured by β-scintillation counting.

Positive control substance(s):

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

Results and discussion

Positive control results:

The current positive control substance hexyl cinnamic aldehyde (25% in acetone:olive oil (4:1 v/v)) produced a stimulation index (SI) of 5.76, thus meeting the reliability criteria for the LLNA (SI > 3).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 25% (v/v): 3.38 50% (v/v): 3.33 100%: 5.18

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

The lymph nodes of all animals per group were pooled and DPM values were measured from the pooled lymph node cell suspensions. Treatment with 0, 25, 50 and 100% test substance in acetone:olive oil (4:1 v/v) resulted in DPM/node per group of 1553.82, 5244.83, 5178.42 and 8048.58, respectively.

Any other information on results incl. tables

CLINICAL OBSERVATIONS:

No mortalities and no signs of systemic toxicity were noted during the study.

 

BODY WEIGHTS:

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the study period.

RESULTS MAIN EXPERIMENT

Table 1. Disintegrations per minute, disintegrations per minute/node and stimulation indices

Test item concentration % (v/v)	Measurement DPM	Calculation		Result
		Number of lymph nodes	DPM per node*	SI	Pos/Neg
0	12430.52	8	1553.82	1.00	n.a.
25	41958.65	8	5244.83	3.38	pos
50	41427.38	8	5178.42	3.33	pos
100	64388.61	8	8048.58	5.18	pos

DPM = disintegrations per minute

* DPM/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

SI = stimulation index of 3.0 or greater indicates a positive result

n.a. = not applicable

Applicant's summary and conclusion

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified