Registration Dossier

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:
28 April 2017 - 14 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
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:
Test Item: Leuco Sulfur Blue 15
Appearance: Dark, near black powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca Ola Hsd mice
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 study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test (4 animals/treatment group, 8 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 12 weeks old (at start of the main test)
Body weight range
at starting: 17.8 – 21.4 g, The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
Acclimatization time: 7 days

Husbandry
Animal health: Only healthy animals (and not showing any sign of skin lesion) were used
Housing during
acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 1 2 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.

The same conditions were used for the dose range finding and main test animals. There were no deviations from these specifications during the experimental phase. The temperature and relative humidity were recorded daily during the acclimatization and experimental phases. Before housing the animals, the microbiological status of the room was checked.

Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive. Relevant batch number, expiry date and contents of the diet are given in Appendix III.
Animals received tap water from watering bottles ad libitum. The drinking water is periodically analysed and is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.

Bedding
Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg (Germany) Holzmühle 1) was available to animals during the study.

Identification of Animals
The individual identification of the animals was performed by numbers on the tail. The cages were marked with identification cards, with information (at least) about study code, species, strain, sex, dose group and individual animal numbers.

Randomization
The animals were set in order of their body weight. The animals were randomly assigned to control and test groups using a randomization scheme. The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the homogeneity and deviations between the groups.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
10 %, 5 %, 2.5 % and 1 % (w/v)
No. of animals per dose:
28 animals/main test (4 animals/treatment group, 8 shared control animals)
Details on study design:
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.
In vivo Treatment

Each mouse was topically treated with 25 µL 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 and 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 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi 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 beta-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-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.

Instrument used for the measurement: 
Name: Tri-Carb 3100TR, Liquid Scintillation Analyzer
Serial Number: 072971

Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index
of 3) was not calculated for the test item.

Interpretation of the Results
The test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The randomization was checked by computer software [SPSS/PC+ (4.0.1)]

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. A significant lymphoproliferative response (SI >= 3) was noted for HCA (SI = 4.8). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
The SI values for all test concentrations were below 3.
Parameter:
SI
Value:
1
Variability:
p = 0.99, r = 0.009
Test group / Remarks:
Test item concentrations of 10 % (w/v).
Remarks on result:
other: no skin sensitization potential in the Local Lymph Node Assay
Parameter:
SI
Value:
1.5
Variability:
p = 0.99, r = 0.009
Test group / Remarks:
Test item concentrations of 5 % (w/v).
Remarks on result:
other: no skin sensitization potential in the Local Lymph Node Assay
Parameter:
SI
Value:
1.1
Variability:
p= 0.99, r= 0.009
Test group / Remarks:
Test item concentration of 2.5% (w/v).
Remarks on result:
other: no skin sensitization potential in the Local Lymph Node Assay
Parameter:
SI
Value:
1
Variability:
p= 0.99, r= 0.009
Test group / Remarks:
Test item concentration of 1% (w/v)
Remarks on result:
other: no skin sensitization potential in the Local Lymph Node Assay
Cellular proliferation data / Observations:
No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 1.0, 1.5, 1.1 and 1.0 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.99, r = 0.009; evaluated by linear regression using SI values).
According to the 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 10 % (w/v) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitizer. No mortality or signs of systemic toxicity were observed during the test. No significant, treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group.

Any other information on results incl. tables

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

Animal

Dose Group

Initial

Terminal

Body Weight

Number

Body Weight

Body Weight

Change

(g)

(g)

(%)

740

Vehicle control

18.2

18.2

0

741

for the positive control:

19.9

20.5

3

742

AOO

19.2

19.4

1

743

 

20.8

20.3

-2

 

Mean

19.5

19.6

0

 

SD

1.1

1.0

 

744

Positive control:

18.8

18.4

-2

745

25 % HCA

18.1

18.5

2

746

in AOO

20.8

21.9

5

747

 

19.8

20.6

4

 

Mean

19.4

19.9

2

 

SD

1.2

1.7

 

768

Vehicle control

20.8

21.6

4

769

for the test item:

17.8

17.6

-1

770

DMSO

19.9

20.3

2

771

 

18.9

18.7

-1

 

Mean

19.4

19.6

1

 

SD

1.3

1.8

 

772

Leuco Sulfur Blue 15

21.4

20.8

-3

773

10 % in DMSO

18.6

19.9

7

774

 

19.3

20.1

4

775

 

20.3

20.8

2

 

Mean

19.9

20.4

3

 

SD

1.2

0.5

 

776

Leuco Sulfur Blue 15

18.6

18.8

1

777

5 % in DMSO

20.1

21.2

5

778

 

19.4

19.3

-1

779

 

21.3

21.7

2

 

Mean

19.9

20.3

2

 

SD

1.1

1.4

 

780

Leuco Sulfur Blue 15

21.3

21.5

1

781

2.5 % in DMSO

18.7

17.2

-8

782

 

20.3

20.7

2

783

 

19.0

19.4

2

 

Mean

19.8

19.7

-1

 

SD

1.2

1.9

 

784

Leuco Sulfur Blue 15

20.2

20.9

3

785

1 % in DMSO

21.3

22.4

5

786

 

19.4

19.1

-2

787

 

18.5

18.4

-1

 

Mean

19.9

20.2

2

 

SD

1.2

1.8

 

HCA =a-Hexylcinnamaldehyde                           AOO = Acetone: Olive oil 4:1 (v/v) mixture

DMSO = Dimethyl sulfoxide                                SD = Standard Deviation

Clinical Observations in the Main Test

Dose Group

Animal
Number

Days

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

Vehicle control for the positive control:
AOO

740

N

N

N

N

N

N

N

N

N

741

N

N

N

N

N

N

N

N

N

742

N

N

N

N

N

N

N

N

N

743

N

N

N

N

N

N

N

N

N

Positive control:

25 % HCA in AOO

744

N

N

N

N

N

N

N

N

N

745

N

N

N

N

N

N

N

N

N

746

N

N

N

N

N

N

N

N

N

747

N

N

N

N

N

N

N

N

N

Vehicle control for the test item:

DMSO

768

N

N

N

N

N

N

N

N

N

769

N

N

N

N

N

N

N

N

N

770

N

N

N

N

N

N

N

N

N

771

N

N

N

N

N

N

N

N

N

Leuco Sulfur Blue 15
10 % in DMSO

772

N

N

N

N

N

N

N

N

N

773

N

N

N

N

N

N

N

N

N

774

N

N

N

N

N

N

N

N

N

775

N

N

N

N

N

N

N

N

N

Leuco Sulfur Blue 15
5 % in DMSO

776

N

N

N

N

N

N

N

N

N

777

N

N

N

N

N

N

N

N

N

778

N

N

N

N

N

N

N

N

N

779

N

N

N

N

N

N

N

N

N

Leuco Sulfur Blue 15
2.5 % in DMSO

780

N

N

N

N

N

N

N

N

N

781

N

N

N

N

N

N

N

N

N

782

N

N

N

N

N

N

N

N

N

783

N

N

N

N

N

N

N

N

N

Leuco Sulfur Blue 15
1 % in DMSO

784

N

N

N

N

N

N

N

N

N

785

N

N

N

N

N

N

N

N

N

786

N

N

N

N

N

N

N

N

N

787

N

N

N

N

N

N

N

N

N

PT = Prior to the treatment

AT = After the treatment

HCA =a-Hexylcinnamaldehyde

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

DMSO = Dimethyl sulfoxide

N = Normal (no symptoms observed)

 


Erythema Scores in the Main Test

Dose Group

Animal Number

Ears

Days

1

2

3

4

5

6

PT

AT

PT

AT

PT

AT

Vehicle control for the positive control:
AOO

740

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

741

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

742

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

743

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Positive control:
25 % HCA in AOO

744

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

745

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

746

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

747

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Vehicle control for the test item:
DMSO

768

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

769

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

770

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

771

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Blue 15
10 % in DMSO

772

L

0

*

0

*

0

*

0

0

0

R

0

*

0

*

0

*

0

0

0

773

L

0

*

0

*

0

*

0

0

0

R

0

*

0

*

0

*

0

0

0

774

L

0

*

0

*

0

*

0

0

0

R

0

*

0

*

0

*

0

0

0

775

L

0

*

0

*

0

*

0

0

0

R

0

*

0

*

0

*

0

0

0

Leuco Sulfur Blue 15
5 % in DMSO

776

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

777

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

778

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

779

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Blue 15
2.5 % in DMSO

780

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

781

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

782

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

783

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

Leuco Sulfur Blue 15
1 % in DMSO

784

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

785

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

786

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

787

L

0

0

0

0

0

0

0

0

0

R

0

0

0

0

0

0

0

0

0

L = Left                                     R = Right                    PT = Prior to treatment                               AT = After the treatment

AOO = Acetone: Olive oil 4:1 (v/v) mixture                    HCA =a-Hexylcinnamaldehyde                  DMSO = Dimethyl sulfoxide

* Dark colour of the formulation made the observation of erythema difficult, but an erythema score < 3 was considered.


Visual Observations of the Lymph Nodes in the Main Test

Dose Group

Animal
Number

Appearance of Lymph Nodes

Vehicle control for the positive control:
AOO

740

N

741

N

742

N

743

N

Positive control:
25 % HCA in AOO

744

Larger than the relevant control (AOO)

745

Larger than the relevant control (AOO)

746

Larger than the relevant control (AOO)

747

Larger than the relevant control (AOO)

Vehicle control for the test item:
DMSO

768

N

769

N

770

N

771

N

Leuco Sulfur Blue 15
10 % in DMSO

772

N

773

N

774

N

775

N

Leuco Sulfur Blue 15
5 % in DMSO

776

N

777

N

778

N

779

N

Leuco Sulfur Blue 15
2.5 % in DMSO

780

N

781

N

782

N

783

N

Leuco Sulfur Blue 15
1 % in DMSO

784

N

785

N

786

N

787

N

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

HCA =a-Hexylcinnamaldehyde

DMSO = Dimethyl sulfoxide

N = Normal


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:

7711

7684.0

1921.0

1.0

AOO

 

 

 

 

Positive control:

36994

36967.0

9241.8

4.8

25 % HCA in AOO

 

 

 

 

Vehicle control for the test item:

5125

5098.0

1274.5

1.0

DMSO

 

 

 

 

Leuco Sulfur Blue 15

4909

4882.0

1220.5

1.0

10 % in DMSO

 

 

 

 

Leuco Sulfur Blue 15

7759

7732.0

1933.0

1.5

5 % in DMSO

 

 

 

 

Leuco Sulfur Blue 15

5475

5448.0

1362.0

1.1

2.5 % in DMSO

 

 

 

 

Leuco Sulfur Blue 15

4960

4933.0

1233.3

1.0

1 % 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= 27.0

# Number of animals/group = 4


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was determined not to have a skin sensitization potential.
Executive summary:

The aim of this study was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. In general, very low solubility of the test item was observed. The maximum concentration producing an adequately stable and homogeneous formulation (suspension/solution) suitable for application on the dorsum of ears of animals was 10 % (w/v) in DMSO using ultrasonic dispersion and stirring. No significant adverse effects (systemic toxicity or irritation) were observed in the DRF up to this maximum concentration. According to this the test item was examined in the main test at concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v). An appropriate positive control (a-Hexylcinnamaldehyde, HCA) and furthermore two negative control groups dosed with DMSO (as vehicle control for the test groups) or AOO (as vehicle control for the positive control group) were employed.

The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation when compared to the concurrent control (SI = 4.8), thus confirming the validity of the assay. No mortality or signs of systemic toxicity were observed during the test. No significant, treatment related effect on the body weights was observed in any dose group. No signs of irritation or any other local effects were observed at the treatment site (ears) in any treatment group. No significantly increased lymphoproliferation (indicated by an SI >= 3) compared to the relevant control (DMSO) was noted for Leuco Sulfur Blue 15 at the applied test concentrations. The observed stimulation index values were 1.0, 1.5, 1.1 and 1.0 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.99, r = 0.009; evaluated by linear regression using SI values). According to the 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 10 % (w/v) as well as the lack of a significant dose-response relationship is considered as evidence that the test item is not a skin sensitizer. Therefore, under the conditions of the present Local Lymph Node Assay the test item was determined not to have a skin sensitization potential.