Corn oil (Zea mays, Gramineae), peroxidised

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 January 2011 to 7 April 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guideline and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

corn oil, peroxidised

IUPAC Name:

corn oil, peroxidised

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 B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 10-11 weeks (main experiment)
- Weight at study initiation: 20.7 +/- 1.3 g
- Housing: in group, in Makrolon Type III cages, with wire mesh top
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 2°C
- Humidity: 45-65%
- Air changes (per hr): no data
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: from 19 January 2011 to 2 February 2011

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0, 25%, 50%, 100%

No. of animals per dose:

4 females per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: a solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100 % of the undiluted test item. Test item solution at different concentrations was prepared using acetone:olive oil (4+1 v/v) as vehicle.
- Irritation:
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 75 (w/v) and 100% once daily each on three consecutive days.
Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch and were immediately pooled per animal and weighed using an analytical balance.
Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value T3 was observed at any observation time and/or if an increase in ear thickness of T25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 25, 50 (w/v), and 100%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: at random
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that 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:
Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25, 50 (w/v), and 100% in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine:
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline (PBS) containing 19.9 µCi of 3HTdR (equivalent to 3HTdR 79.6 µCi/mL) were injected into each test and control mouse via the tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: at least once daily from experimental start to necropsy.
- Body weights: in the pre-test prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.
- Ear thickness: in the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).
- Ear weights: in the pre-test after sacrifice; biopsy punches were taken from each ear.
- Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

not applicable

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice. The periodic positive control experiment was performed with alpha-Hexyl cinnamaldehyde in acetone:olive oil (4+1 v/v) using CBA/CaOlaHsd mice in December 2010.
SI = 1.00, 0.85, 1.85 and 7.19, respectively at 0%, 5%, 10% and25%
EC3 = 13.2% (w/v)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1.00, 1.43, 2.36 and 1.84 at respectively 0, 25%, 50% and 100%

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See table

Any other information on results incl. tables

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG a)	Number of lymph nodes	DPM per lymph node b)	S.I.
-	BG I	15	-	-	-	-
-	BG 2	14	-	-	-	-
0	1	3470	3456	8	431.9	1.00
25	2	4936	4936	8	616.9	1.43
50	3	8157	8143	8	1017.8	2.36
100	4	6370	6356	8	794.4	1.84

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Other observations:

- Viability / Mortality: no deaths occurred during the study period.

- Clinical Signs: no symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

- Body Weights: the body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item peroxidised corn oil was not a skin sensitiser under the test conditions of the study.

Executive summary:

In a study according to OECD TG 429 and GLP, the test item peroxidised corn oil dissolved in acetone:olive oil (4+1 v/v) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50 (w/v), and 100%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. In this study Stimulation Indices (S.I.) of 1.43, 2.36, and 1.84 were determined with the test item at concentrations of 25, 50 (w/v), and 100% in acetone:olive oil (4+1 v/v), respectively.

The test item peroxidised corn oil was not a skin sensitiser under the test conditions of this study.