Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 09 August 2011 and 25 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline
Justification for type of information:
Study pre-dates the introduction of the in vitro methods.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: amber coloured liquid
- Lot/batch No.: 0100954340

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid floor polypropylene cages furnished with softwood flakes.
- Diet (e.g. ad libitum): 2014C Teklad global rodent diet, ad libitum
- Water (e.g. ad libitum):mains tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle


IN-LIFE DATES: No data available

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1st study: 25%, 50%, 100%
2nd study: 2.5%, 10%, 25%
No. of animals per dose:
5 mice per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the substance was considered suitable for dosing in acetone/olive oil (4:1)at a concentration of 50% as upon mixing by vortex mixer the formulation formed a solution which could be administered by micropipette.
- Irritation: mild
- Lymph node proliferation response: Expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ration of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: “Skin Sensitisation: Local Lymph Node Assay” / “Skin Sensitisation (Local Lymph Node Assay of Commission directive 2004/73/EC”
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ^3HTdR incorporation compared to control values.


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. (50%)
Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guideline.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was interpreted using GraphPad PRISM version 3.03.

Results and discussion

Positive control results:
Concentration v/v in acetone/olive oil 4:1: 25%
Stimulation Index: 9.61
Result: Positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Conc. (% v/v) S.I. Test 1 Vehicle NA 25% 6.32 50% 10.65 100% 16.91 Test 2 Vehicle NA 2.5 1.31 10 1.59 25 6.59 Positive control 9.61
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (% v/v) Mean dpm/Node Test 1 Vehicle 1417.13 25% 8952.87 50% 15086.68 100% 23957.95 Test 2 Vehicle 1411.25 2.5 1889.71 10 2299.81 25 9512.12 Positive control 13870.04

Any other information on results incl. tables

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in

Acetone/olive oil 4:1

dpm

Stimulation Indexb

 

Result

 Test 1      
 Vehicle Control  1417.13  NA NA 
 25  8952.87  6.32  Positive
 50  15086.68  10.65  Positive
 100  23957.95  16.91  Positive
 Test 2      
Vehicle  1411.25  NA NA 
 2.5  1889.71  1.31  Negative
 10  2299.81  1.59  Negative
 25  9512.12  6.59  Positive
 Positive Control  13870.04  9.61  Positive

 

Dpm = disintegrations per minutes

b = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not Applicable

 

Individual clinical Observations and Mortality Data

 Mild redness on the ears was noted in animals treated with the undiluted test item. There were no deaths.  No signs of systemic toxicity were noted in the test or control animals during the test.

 

Individual Bodyweights and Bodyweight Changes

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

 

EC3:

The results of the statistical analysis of the data indicated there was no significant difference between the vehicle control group and the test item groups. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be14%.

 

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
In a Local Lymph Node Assay in the Mouse, the substance initiated a Stimulation Index of greater than 3 at concentrations of 100, 50 and 25% and EC3 was determined to be 14.2%; thus is considered to be a skin sensitizer under the conditions of the test.
Executive summary:

Introduction.A study was performed to assess the skin sensitization potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements OECD 429 and Method B42.

Methods.Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.

(1)  Test 1: three groups, each of five animals, were treated with 50 μl (25 μl per ear) of the test material undiluted or as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% or 10% w/w;A further group of five animals was treated with acetone/olive oil 4:1 alone. The pool method was used;

(2)  Test 2: three groups, each of five animals, were treated with the test item at concentrations of 25%, 10% and 2.5% v/v in acetone/olive oil 4:1.    An  additional  group  of five animals  was  treated  with acetone/olive oil 4:1 alone.   A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, a-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

 

Results.The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

 

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

 

Concentration (% w/w) in

Acetone/olive oil 4:1

dpm

Stimulation Indexb

 

Result

 Test 1

 

 

 

 Vehicle Control

 1417.13

 NA

NA 

 25

 8952.87

 6.32

 Positive

 50

 15086.68

 10.65

 Positive

 100

 23957.95

 16.91

 Positive

 Test 2

 

 

 

Vehicle

 1411.25

 NA

NA 

 2.5

 1889.71

 1.31

 Negative

 10

 2299.81

 1.59

 Negative

 25

 9512.12

 6.59

 Positive

 Positive Control

 13870.04

 9.61

 Positive

 

 

 

According to the calculation the concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was found to be 14%.

 

 

Conclusion.The test material was considered to be a sensitiser under the conditions of the test.