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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 January 2010 - 03 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with international guidelines and to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Oxooil LS9

- Substance type: Industrial material
- Physical state: liquid
- Analytical purity: 95.3%
- Purity test date: Not reported
- Lot/batch No.: 13.06.2008 0649/82531
- Expiration date of the lot/batch: 8 June 2010
- Stability under test conditions: Not stated
- Storage condition of test material: Refrigerated, 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 UK Ltd.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 18.5 to 20.8 g
- Housing: Individually in polycarbonate cages with woodflake bedding, Nestlets and mouse houses for environmental enrichment.
- Diet (e.g. ad libitum): standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 19 January 2010 To: 02 February 2010

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 and 50% v/v with the vehicle
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
- Irritation: Mouse dosed with 25 µL of test substance (50% v/v) to the dorsal surface of each ear to 3 consecutive days. No signs of irritation. This dose chosen as maxiumum dose for the main study.
- Lymph node proliferation response: Not investigated

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: if at least one concentration of the test substance results in a three-fold greater increase in 3H-methyl thymidine incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:

Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 μl of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was appled to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear usingthe tip of the pipette. In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 50 μl of phosphate buffered saline containing 3H-methyl Thymidine (3HTdR: 80 μCi/mL) giving a nominal 20 μCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.
In the main phase of the study, five hours following the administration of 3HTdR on Day 6 all mice were humanely killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group.
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5%) following the final wash.
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5% TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by β-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
A study was performed to confirm the sensitivity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. The study was performed using the murine local lymph node assay (OECD 429 individual animal approach) and a known moderate sensitizer – hexyl cinnamic aldehyde (HCA). This study was conducted between the 14 and 20 October 2009 using ten mice of the
CBA/ca strain. The positive control study is considered to be valid if the results from the HCA group have a three-fold greater increase in 3HTdR incorporation compared to control values. In this assay the test/control ratio obtained for HCA at 25% was 6.0. This indicates that HCA demonstrates the potential to induce skin sensitization (delayed contact hypersensitivity) and confirms the sensitivity of the technique to detect sensitization potential.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
5 (50 % v/v)
Parameter:
SI
Value:
1.4
Test group / Remarks:
4 (25 % v/v)
Parameter:
SI
Value:
0.8
Test group / Remarks:
3 (10 % v/v)

Any other information on results incl. tables

Table 1 Group dpm/node and test/control ratios

 

Group

Concentration

% v/v

Group mean

dpm/node

Number of lymph nodes per group

dpm/node

test/control

ratio†(SI)

Classification

+ = positive

- = negative

2

AOO

6121.60

8.0

759.32

n/a

n/a

3

10

4936.20

8.0

611.14

0.8

-

4

25

8534.30

8.0

1060.91

1.4

-

5

50

28683.60

8.0

3579.57

4.7

+

AOO     Acetone:olive oil (4:1 v/v)

          Test/control ratio of greater than 3.0 indicates a positive result

dpm       Disintegrations per minute (Less mean background count of 47.05 dpm)

n/a         Not applicable

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
As a SI of 3 or more was recorded for highest concentration tested, Oxooil LS9 was considered to have the potential to cause skin sensitization (delayed contact hypersensitivity). The EC3 was calculated and determined to be 37% v/v.
Executive summary:

A study was performed at the Laboratories of Huntingdon Life Sciences, Alconbury, UK, on behalf of Evonik Oxeno GmbH. , to assess the skin sensitisation potential of Oxooil LS9 using the murine local lymph node assay. The study was conducted to GLP and in accordance with OECD Guideline 429 and EU Method B.42. Three groups of four mice were treated by daily application to the dorsal surface of both ears for three consecutive days with 25 µl of 10%, 25% or 50% Oxooil LS9 in 4:1 v/v acetone:olive oil. A similarly constituted control group received vehicle alone. The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio). The test substance is regarded as a sensitizer if at least one concentration of the chemical results in a three-fold greater increase in 3HTdR incorporation compared to control values.

In this assay the test/control ratios obtained for 10, 25 and 50% v/v were 0.8, 1.4 and 4.7 respectively which indicates that Oxooil LS9 showed the potential to induce skin sensitization. The EC3 was calculated and determined to be 37% v/v.