Fatty acids, safflower-oil, Et esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-06-07 - 2010-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, safflower-oil, Et esters

EC Number:

293-106-3

EC Name:

Fatty acids, safflower-oil, Et esters

Cas Number:

91051-53-5

IUPAC Name:

Fatty acids, safflower-oil, Et esters

Test material form:

other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: approx. 10 weeks
- Housing: individually
- Diet: ad libitum (SM R/M-Z from SSNIFF® Spezialdiaeten GmbH, Soest, Germany)
- Water: ad libitum (tap water)
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.9 -22.9
- Humidity (%): 42-78
- 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:

0, 25, 50, 100 % (w/w)

No. of animals per dose:

5

Details on study design:

PRELIMINARY IRRITATION TEST:
- Irritation: Slight erythema was observed at 50 and 100%. No signs of systemic toxicity were observed in any of the animals examined. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
INDUCTION
The dorsal surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that the vehicle was administered instead of the test substance.

EXCISION OF LYMPH NODES
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 uCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

TISSUE PROCESSING FOR RADIOACTIVITY
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 urn). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

RADIOACTIVITY MEASUREMENTS
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

OBSERVATIONS
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of alt other (local) effects were recorded.

Positive control substance(s):

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

Statistics:

Observations/measurements in the study were recorded electronically using the following programme(s): REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ, USA); Quantasmart 2.03 (PerkinElmer Life Sciences, Boston, MA, USA): LSC software

Results and discussion

Positive control results:

The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 25, 50 and 100 % were 2.3, 6.6 and 8.2 respectively. An EC3 value of 29.1 % was calculated.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100 % were 1356, 3986 and 4909 DPM respectively. The mean DPM/animal value for the vehicle control group was 602 DPM.

Results 3

Parameter:

SI

Value:

2.3

Test group / Remarks:

Test Group 2 (animal 6,7,8,9,10): Test Substance 25% (w/w)

Results 4

Parameter:

SI

Value:

6.6

Test group / Remarks:

Test Group 3 (animal 11,12,13,14,15): Test Subtsance 50% (w/w)

Results 5

Parameter:

SI

Value:

8.2

Test group / Remarks:

Test Group 4 (animal 16,17,18,19,20): test Substance 100 % (w/w)

Any other information on results incl. tables

Preliminary Irritation study

Slight erythema was observed at 50 and 100%. No signs of systemic toxicity were observed in any of the animals examined. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Main study

The slight irritation of the ears as shown by most animals treated at 50 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

The auricular lymph nodes of the animals at 50 and 100% appeared larger in size as compared to the other treated groups. The auricular lymph nodes of the animals at 25% were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in several animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test substance was a skin sensitizer.

Executive summary:

The study was carried out on the basis of the OECD guideline No. 429. The test substance concentrations seleted for the main study were based on the results of a preliminary study.

In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v). Three days after the last exposure, all animals were injected with 3H-methyt thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The slight irritation of the ears as shown by most animals treated at 50 and 100% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined. The auricular lymph nodes of the animals at 50 and 100% appeared larger in size when compared to the other treated groups. The auricular lymph nodes of the animals at 25% were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in

any of the animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 1356, 3986 and 4909 DPM respectively.

The mean DPM/animal value for the vehide control group was 602 DPM.

The SI values calculated for the substance concentrations 25, 50 and 100% were 2.3, 6.6 and

8.2 respectively.

These results indicate that the test substance could elicit an SI ~ 3. The data showed a dose-response relationship and an EC3 value (the estimated test substance concentration that will give a SI =3) of 29.1% was calculated.

The six-month reliability check with alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

According to the recommendations made in the test guidelines, the test item would be regarded as skin sensitizer, based on these results.