Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 03-August 22, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (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
Test material form:
solid: particulate/powder
Details on test material:
Batch No.: UGe-RS Kilo 1
Color: Yellow
Odor: Not specific
Storage conditions: room temperature
Solubility and stability: Very good solubility in water, stable in solution at least 2 days
Expiring date: 12.12.2018
Correction factor: 1.07
Specific details on test material used for the study:
Test item: Yellow LF 6911
Batch No.: UGe-RS Kilo 1
Appearance: Yellow powder
Expiration date: 12 December 2018
Storage: Room temperature
Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) were applied to assure personnel health and safety.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during the test: Good conventional
Number of animals: 28 animals/main test (4 animals/treatment group)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 10 weeks old (at start of the main test)
Body weight range at starting:16.6 – 21.4 g
The weight variation in animals involved in the study did not exceed  20 % of the mean weight.
Acclimatization time: 7 days
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
In-life phase: June 15- June 21, 2016


Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
25 %, 10 %, 5 % or 2.5 % (w/v)
No. of animals per dose:
4 animals/treatment group
Details on study design:
Based on the preliminary test results the maximum attainable concentration (based on solubility) of 25 % (w/v) was used in the main test. The test item was tested also at three additional, lower concentrations (10 %, 5 % and 2.5 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.
Animals in the treatment groups were treated with the negative (vehicle) controls (DMSO or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the dose range finding test.
Table 2. Criteria for Erythema Scores

Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar
formation preventing grading of erythema 4

Groups Test item concentration(% w/v) Positive control
concentration (% w/v) No. of animals

1 Vehicle control for the positive control: AOO - - 4
2 Positive control: HCA in AOO - 25 4
3 Vehicle control for the test item: DMSO - - 4
4 Yellow LF 6911 in DMSO 25 - 4
5 Yellow LF 6911 in DMSO 10 - 4
6 Yellow LF 6911 in DMSO 5 - 4
7 Yellow LF 6911 in DMSO 2.5 - 4

In vivo Treatment
Each mouse was topically treated with 25 L of the appropriate formulations of the test item, the positive control substance or the vehicles (see Table 3) using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 L of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 microCi# of
3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the -scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The -counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.






Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Randomization based on body weigt was checked by SPSS PC+
Calculation of stimulation indices were made by EXCEL software

Results and discussion

Positive control results:
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI  3) was noted for HCA (SI = 11.9). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

In vivo (LLNA)

Results
Key result
Parameter:
SI
Value:
>= 3
Variability:
r = 0.56, p = 0.44
Test group / Remarks:
SI values were 1.2, 1.2, 2.6 and 1.6 at 25 %, 10 %, 5 % and 2.5 % (w/v) concentrations
Cellular proliferation data / Observations:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score  3) or any other local effect were observed in any treatment group.
No significant, treatment related effect on body weights was observed during the test. Body weight, decreased by > 5 % was observed in the AOO treatment group only (1/4 animals, 7 % decrease) but it was considered neither significant nor treatment related.
No significant lymphoproliferative response (SI  3) compared to the relevant control (DMSO) was noted for Yellow LF 6911 at the applied test concentrations. The observed stimulation index values were 1.2, 1.2, 2.6 and 1.6 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively.
Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.44, r = 0.56).

Any other information on results incl. tables

Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test:

Animal

Dose Group

Initial

Terminal

Body Weight

Number

 

Body Weight

Body Weight

Change

 

 

(g)

(g)

(%)

52

Vehicle control for the positive control:

16.6

16.5

-1

53

AOO

19.8

18.5

-7

54

 

20.3

21.5

6

55

 

18.0

20.0

11

 

Mean

18.7

19.1

2

 

SD

1.7

2.1

 

56

Positive control:

18.3

18.8

3

57

25 % HCA

20.2

20.7

2

58

 in AOO

17.7

18.2

3

59

 

20.8

20.6

-1

 

Mean

19.3

19.6

2

 

SD

1.5

1.3

 

60

Vehicle control for the test item:

20.0

21.4

7

61

DMSO

21.3

20.8

-2

62

 

18.3

17.8

-3

63

 

17.8

18.3

3

 

Mean

19.4

19.6

1

 

SD

1.6

1.8

 

64

Yellow LF 6911

18.1

18.8

4

65

25 %

17.5

18.4

5

66

in DMSO

20.6

21.1

2

67

 

20.1

20.8

3

 

Mean

19.1

19.8

4

 

SD

1.5

1.4

 

68

Yellow LF 6911

18.8

18.3

-3

69

10 %

21.2

22.9

8

70

in DMSO

19.3

19.2

-1

71

 

17.3

17.7

2

 

Mean

19.2

19.5

2

 

SD

1.6

2.3

 

72

Yellow LF 6911

19.5

19.9

2

73

5 %

18.0

17.8

-1

74

in DMSO

20.6

20.9

1

75

 

17.5

17.2

-2

 

Mean

18.9

19.0

0

 

SD

1.4

1.7

 

76

Yellow LF 6911

21.4

22.4

5

77

2.5 %

19.0

19.3

2

78

in DMSO

17.7

17.7

0

79

 

18.1

19.2

6

 

Mean

19.1

19.7

3

 

SD

1.7

2.0

 

 

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMSO = Dimethyl sulfoxide

Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test:

Dose Group

Animal

Ears

Day 1*

Day 3$

Day 3

Day 6#

Day 6

 

Number

value (mm)

value (mm)

% deviation

value (mm)

% deviation

 

970

L

0.21

0.22

4.8

0.24

14.3

Yellow LF 6911

 

R

0.21

0.21

0.0

0.24

14.3

25 % in DMSO

984

L

0.21

0.21

0.0

0.23

9.5

 

 

R

0.21

0.21

0.0

0.23

9.5

 

971

L

0.21

0.22

4.8

0.25

19.0

Yellow LF 6911

 

R

0.21

0.22

4.8

0.24

14.3

10 % in DMSO

48

L

0.21

0.23

9.5

0.23

9.5

 

 

R

0.21

0.23

9.5

0.23

9.5

L = Left                          R = Right

DMSO = Dimethyl sulfoxide

* Ear thickness was measured prior to the first treatment.

$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).

# Ear thickness was measured at the end of the test.

DPM and Stimulation Index Values for all Groups in the Main Test:

Dose Group

Measured

Group*

DPM/Mouse#

Stimulation

 

DPM/group

DPM

 

Index Values

Vehicle control for the positive control:

4258

4229.5

1057.4

1.0

AOO

 

 

 

 

Positive control:

50157

50128.5

12532.1

11.9

25 % HCA in AOO

 

 

 

 

Vehicle control for the test item:

3607

3578.5

894.6

1.0

DMSO

 

 

 

 

Yellow LF 6911

4286

4257.5

1064.4

1.2

25 % in DMSO

 

 

 

 

Yellow LF 6911

4265

4236.5

1059.1

1.2

10 % in DMSO

 

 

 

 

Yellow LF 6911

9293

9264.5

2316.1

2.6

5 % in DMSO

 

 

 

 

Yellow LF 6911

5662

5633.5

1408.4

1.6

2.5 % in DMSO

 

 

 

 

 

HCA =a-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMSO = Dimethyl sulfoxide

 

*Group DPM = measured DPMgroup- average DPMbackground

Average DPMbackground= 28.5

# Number of animals/group = 4

Applicant's summary and conclusion

Conclusions:
Under the conditions of the present assay, Yellow LF 6911 tested at the maximum feasible concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMSO) was shown to have no skin sensitization potential in the Local Lymph Node Assay.
Executive summary:

Since no failed treatment, sign of systemic toxicity or irritation was observed during the test no treatment group was excluded from the evaluation. Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMSO) and in the test item treated groups. No significant lymphoproliferative response (SI >=3) compared to the relevant control (DMSO) was noted for Yellow LF 6911at the applied test concentrations. The observed stimulation index values were 1.2, 1.2, 2.6 and 1.6 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively.

Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.44, r = 0.56).

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score³ 3) or any other local effect were observed in any treatment group.

No significant, treatment related effect on body weights was observed during the test. Body weight, decreased by > 5 % was observed in the AOO treatment group only (1/4 animals, 7 % decrease) but it was considered neither significant nor treatment related.

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation(indicated by an SI>= 3)up to the maximum attainable concentration of 25 % (w/v, based on solubility) and also the lack of a significant
dose-response relationship are considered evidence that Yellow LF 6911
is not a skin sensitizer.