strain mouse following topical application to the dorsal surface of the ear. This study was 

designed to be compatible with the procedures indicated by the following internationally · 

accepted guidelines and recommendations: 

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local 

Lymph Node Assay" (adopted 22 July 2010) 

• Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation 

(EC) No. 440/2008 

The assay has undergone extensive inter-laboratory validation and has been shown to reliably 

detect test items that are moderate to strong sensitizers. 

Experimental Starting Date: 

Experimental Completion Date: 

18 June 2014 

08 July 2014 

Test item characterization data are the responsibility of the Sponsor. 

All raw data will be retained in L..jc_s_t _______________ __. 

METHODS 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a 

concentration of 1 00%, this concentration was selected as the highest dose investigated in the 

main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated 

with 50 J.lL (25 J.lL per ear) of the undiluted test item or the test item as a solution in dimethyl 

sulphoxide at concentrations of 50% or 25% v/v. A further group of four animals was treated 

with dimethyl sulphoxide alone. 
Main Test 

The radioactive disintegrations per minute per lymph node and the stimulation index are given in 

Table 4. Individual clinical observations and mortality data for test and control animals are 

given in Table 5 and the individual body weights and body weight change for test and control 

animals are given in Table 6. 

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: 

Concentration (% v/v) in Stimulation Index Result dimethyl sulphoxide 

25 1.54 Negative 

50 1.84 Negative 

100 1.85 Negative 

CONCLUSION 

The test item was considered to be a non-sensitizer under the conditions of the test. 

This study was conducted in a facility operating to Good Laboratory Practice within the UK 

national GLP monitoring programme, but the study report has not been audited by the QA Unit. 

No formal claim of GLP compliance is made for this study. This report is an accurate record of 

the study and its outcome. 

A Pooles 

Study Director 

Date: 
1 6 Dose Dose Dose Dose Dose Dose 

100% S-1 17 16 0 0 0 0 0 0 0 0 0 

0 = No signs of systemic toxicity 
0 0 0 0 0 0 0 0 0 0 0 0 
overall mean (mm) 0.21 0.23 0.21 

overall mean ear thickness 9.76 2.44 

change(%) na 

na == Not applicable 
25 41186.34 5148.29 1.54 Negative 

50 49302.47 6162.81 1.84 Negative 

100 49580.07 6197.51 1.85 Negative 

dpm = Disintegrations per minute 

a= Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total 

number oflymph nodes) 

b = Stimulation Index of3.0 or greater indicates a positive result 

na = Not applicable 
Vehicle 

0 0 0 0 0 0 0 0 0 

l-4 0 0 0 0 0 0 0 0 0 

0 0 0 0 0 0 0 0 0 

2-2 0 0 0 0 0 0 0 0 0 

25 

2-3 0 0 0 0 0 0 0 0 0 2-4 0 0 0 0 0 0 0 0 0 

0 0 0 0 OFI OFI OFI OF! OF! 

3-2 0 0 0 0 OFI OF! OFI OFI OFI 

50 

0 0 0 0 OF! OF! OFI OFI OFI 

3.4 0 0 0 0 OF! OF! OF! OF! OFI 

0 0 0 0 OF! OFI OF! OFI OFI 

0 0 0 0 OF! OF! OF! OF! OF! 

100 

4-3 0 0 0 0 OF! OF! OFI OF! OFI 

4-4 0 0 0 0 OF! OFI OFI OF! OFI 

0 = No signs of systemic toxicity 

Fl = Fur loss 
Vehicle 

1-3 21 19 -2 

1-4 19 17 - 2 

2-1 19 21 2 

2-2 18 18 0 

25 

2-3 18 19 1 2-4 20 20 0 

3-1 19 19 0 3-2 18 19 1 

50 

3-3 18 17 - 1 

3.4 18 19 1 

4-1 18 19 1 

4-2 18 20 2 

100 

4-3 19 19 0 4-4 18 18 0