O,O'-dioctadecylpentaerythritol bis(phosphite)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 February 2013 to 05 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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:

O,O'-dioctadecylpentaerythritol bis(phosphite)

EC Number:

223-276-6

EC Name:

O,O'-dioctadecylpentaerythritol bis(phosphite)

Cas Number:

3806-34-6

Molecular formula:

C41H82O6P2

IUPAC Name:

3,9-bis(octadecyloxy)-2,4,8,10-tetraoxa-3,9-diphosphaspiro[5.5]undecane

Test material form:

other: granular

Details on test material:

- Physical state: White granular solid
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

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
- Weight at study initiation: 15 to 23 g
- Housing: Individually in suspended solid-floor polypropylene cages furnished with soft wood flakes
- Diet: ad libitum, 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon UK)
- Water: ad libitum, mains tap water
- Acclimation period: A minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: Approximately 15 changes per hour
- Photoperiod: 12 hours light/12 hours dark

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2.5, 5 or 10% w/v in acetone/olive oil 4:1

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The vehicle was selected as it provided the highest concentration suitable for dosing
- Irritation: A preliminary screening test for toxicity and irritation was performed in one mouse prior to the main test. The mouse was treated by daily application (for three consecutive days) of the test material to the dorsal surface of each ear at a concentration of 10% w/v in acetone/olive oil 4:1 at a volume of 25 µL per ear. The mouse was observed twice daily on days 1, 2 and 3 and then once daily thereafter until day 6. Local skin irritation was scored daily as erythema. Any clinical signs of toxicity were also recorded. The thickness of each ear was measured using a Mitutoyo 547-300S digital thickness gauge pre-dose on day 1 post dose on day 3 and on day 6. Mean ear thickness was calculated between time days 1 and 3 and days 1 and 6. A mean ear thickness increase of 25% or more was considered to indicate excessive irritation and limited biological relevance to the determination of sensitisation. The preliminary test did not indicate any signs of local irritation or systemic toxicity of the test material, therefore 10% w/v in acetone/olive oil 4:1 was selected as the highest dose for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Animal assignment: Random
- Criteria used to consider a positive response: The test material would be considered a skin sensitiser if it produced in at least one concentration an increase of threefold or more of ³HTdR incorporation (Stimulation Index) compared to the control.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test material in suspension was administered daily for three consecutive days to the dorsal surface of each ear at doses of 2.5, 5 or 10% w/v in acetone/corn oil 4:1. Four animals were treated per test material concentration, a further four animals were included as a vehicle control. Each treatment was spread evenly over the dorsal surface of each ear. Five days following the first topical application of the test material suspension, each mouse was administered 250 µL of ³H-methyl thymidine (80 µCi/mL, specific activity 2.0 Ci/mmol) in phosphate buffered saline intravenously via the tail vein, giving a total dose of 20 µCi per mouse. Five hours post administration of ³HTdR, the animals were sacrificed and the draining lymph nodes for all four mice per group were excised and pooled.

A single cell suspension of the pooled lymph nodes was prepared by gentle mechanical disaggregation using a 200-mesh stainless steel gauze. The lymph nodes were rinsed with 4 mL of phosphate buffered saline (PBS) into a petri dish. The lymph node suspension was then transferred into a centrifuge tube and the remaining lymph node cells were washed from the petri dish into the tube with an additional 5 mL of PBS. The suspension was centrifuged at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL PBS and re-pelleted. The pellet was then resuspended in 3 mL of 5% Trichloroacetic acid (TCA) to precipitate out the radioactive material.

After 18 hours of incubation at 4°C, the precipitates were recovered by centrifugation (2100 rpm, approximately 450 g for 10 minutes). The pellet was resuspended in 1 mL TCA and transferred to 10 mL of scintillation fluid. The vials containing the samples were placed in the sample changer of the scintillator and left for 20 minutes to reduce the risk of luminescence. Prior to counting the samples were shaken vigorously. The ³HTdR incorporation was measured by β-scintillation counting. The disintegrations per minute were measured using a Beckman LS6500 scintillation system.

Positive control substance(s):

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

other: phenylacetaldehyde (CAS No 122-78-1) Tested at 2.5% in propylene glycol

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 2.5% w/w = 1.16 5% w/w = 1.58 10% w/w = 2.80

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle = 11110.37 dpm (1388.80 dpm/Node) 2.5% w/w = 12860.08 dpm (1607.51 dpm/Node) 5% w/w = 17532.82 dpm (2191.60 dpm/Node) 10% w/w = 31117.88 dpm (3889.74 dpm/Node)

Any other information on results incl. tables

Table 2: Clinical signs*, mortality and bodyweights

Concentration (% w/w)	Animal Number	Day 1			Day 2		Day 3		Day 4	Day 5	Day 6		Bodyweight Change (g)
		Mortality		Bodyweight (g)	Mortality		Mortality
		A	B		A	B	A	B			Mortality	Bodyweight (g)
Vehicle	1-1	0	0	21	0	0	0	0	0	0	0	22	1
	1-2	0	0	21	0	0	0	0	0	0	0	20	-1
	1-3	0	0	20	0	0	0	0	0	0	0	20	0
	1-4	0	0	23	0	0	0	0	0	0	0	21	-2
2.5	2-1	0	0	23	0	0	0	0	0	0	0	21	-2
	2-2	0	0	19	0	0	0	0	0	0	0	19	0
	2-3	0	0	19	0	0	0	0	0	0	0	19	0
	2-4	0	0	19	0	0	0	0	0	0	0	20	1
5	3-1	0	0	21	0	0	0	0	0	0	0	22	1
	3-2	0	0	19	0	0	0	0	0	0	0	20	1
	3-3	0	0	21	0	0	0	0	0	0	0	20	-1
	3-4	0	0	19	0	0	0	0	0	0	0	20	1
10	4-1	0	0	19	0	0	0	0	0	0	0	20	1
	4-2	0	0	18	0	0	0	0	0	0	0	20	2
	4-3	0	0	23	0	0	0	0	0	0	0	23	0
	4-4	0	0	21	0	0	0	0	0	0	0	21	0
A = Pre-dose B = Post dose * No symptoms of toxicity were observed during the test

 

Table 3: Disintegrations per minute, disintegrations per minute/node and stimulation index

Concentration (% w/w)	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	11110.37	1388.80	na	na
2.5	12860.08	1607.51	1.16	Negative
5	17532.82	2191.60	1.58	Negative
10	31117.88	3889.74	2.80	Negative
dpm = disintegrations per minute ^adisintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes) ^bStimulation index of 3.0 or greater indicates a positive result na = not applicable

Validation of the results:

The results of the study were compared to the responses of known sensitisers and non-sensitisers from the laboratory's in-house validation. The results of the routine positive control studies demonstrated that the stain of mouse used in the study from the performing laboratory produce satisfactory responses using known sensitisers and non-sensitiser. The results of the current study are therefore considered to be adequate for the prediction of the sensitising potential of the test material.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, the test material was found to be a non-sensitiser when applied to the skin of female CBA mice.

Executive summary:

The study was performed to assess the skin sensitisation potential of the test material to female CBA mice following topical application to the dorsal surface of the ear. The study was GLP compliant and performed to the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600. The test material was screened for irritancy and systemic toxicity at 10% w/v in acetone/corn oil 4:1. This was selected as the highest dose tested in the main test. 25 µL of the test material in suspension was administered daily for three consecutive days to the dorsal surface of each ear at doses of 2.5, 5 or 10% w/v in acetone/corn oil 4:1. Four animals were treated per test material concentration, a further four animals were included as a vehicle control. Five days following the first topical application of the test material suspension, each mouse was administered 250 µL of ³H-methyl thymidine (80 µCi/mL, specific activity 2.0 Ci/mmol) in phosphate buffered saline intravenously via the tail vein, giving a total dose of 20 µCi per mouse. Five hours post administration of ³HTdR, the animals were sacrificed and the draining lymph nodes for all four mice per group were excised and pooled. The lymph nodes were prepared, and the ³HTdR incorporation was measured by β-scintillation counting. The scintillation index was calculated as the mean radioactive incorporation for each treatment group divided by the mean radioactivity incorporation of the vehicle control group. The stimulation indices of the test material at 2.5, 5 and 10% w/v in acetone/olive oil 4:1 were 1.16, 1.58 and 2.80 respectively. A stimulation index indicating a threefold increase of ³HTdR incorporation above the respective vehicle control was considered to be positive. Under the conditions of the test, the test material when tested up to 10 w/v in acetone/olive oil 4:1 was found to be a non-sensitser to the skin of CBA female mice.