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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 Sep - 11 Oct 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
EC Number:
227-030-9
EC Name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
Cas Number:
5610-94-6
Molecular formula:
C₄₃H₂₂N₆O₁₃S₃
IUPAC Name:
4-benzoylbenzene-1,2,3-triyl tris(6-diazo-5,6-dihydro-5-oxonaphthalene-1-sulphonate)
Test material form:
solid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: grinding of the test item in a mortar to reduce the particle size for increased skin contact

FORM AS APPLIED IN THE TEST
liquid (1 tablet disolved in 200 mL deionized water)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
Ola Hsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Horst, the Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 12 weeks (pre-screen test), 11- 12 weeks (main study)
- Weight at study initiation: 20.9 - 21.8 g (pre-screen test), 19.3 - 20.3 g (main study) (range)
- Housing: 2 - 4 animals per cage, in Makrolon Type II (pre-screen test) / III (main study), with wire mesh top and granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 45 – 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
(PG)
Concentration:
5, 10 and 20%
No. of animals per dose:
2 females (pre-screen test)
4 females (main test)
Details on study design:
PRE-SCREEN TESTS:
Two concentrations (10 and 20%, dissolved in PG) were applied topically to the ears of each animal (one mouse per concentration), once a day for 3 consecutive days. Animals were observed for clinical signs of systemic toxicity and local irritation at the application site at least once daily. Furthermore, body weights and ear thickness measurements were performed (body weight: before initial application and on Day 6, ear thickness: before initial application, on Day 6 and on Day 3). The ears were punched after sacrifice (Day 6) at the apical area using a biopsy punch (Ø 8 mm, corresponding to 0.5 cm2), pooled per animal and weighed using an analytical balance. None of the animals showed any signs of systemic toxicity or significant irritation (defined as an erythema score ≥ 3 and /or an increase of more than 25% in ear thickness). No mortality was observed.
Based on these results and considering that 20% was the maximum attainable concentration, 20 % (w/v) was selected as the highest test concentration for the main study. This concentration was expected not to induce systemic toxicity, nor to induce an increase in ear thickness exceeding 25% or to induce dermal erythema with a score of 3 or more.

- Compound solubility: 20% (maximum attainable concentration due to solubility properties)
- Irritation: no irritation was observed
- Systemic toxicity: no systemic toxicity was observed
- Ear thickness measurements: yes (less than 25% increase in ear thickness was measured)
- Erythema scores: 0 (both treatment groups)

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine (3HTdR) incorporation, determined by ß-scintillation
- Criteria used to consider a positive response: SI ≥ 3

TREATMENT PREPARATION AND ADMINISTRATION:
25 μl of each dose formulation (5, 10 and 20%) or the vehicle alone were applied to the dorsal skin of each ear of each animal once a day for 3 consecutive days. On Day 6, 20 μCi 3H-methyl thymidine, contained in 250 μL of PBS (= 80.1 μCi/mL), was administered to each mouse via the tail vein. Approximately 5 h after administration, local lymph nodes were collected, pooled and separated by gentle mechanical disaggregation through a stainless steel gauze (200 μm mesh size). After washing two times in PBS the lymph node cells were treated with ~3 mL of 5% trichloroacetic acid (TCA) at ~4 °C for at least 18 h before the determination of the amount of 3H-methyl thymidine incorporation (as disintegrations per minutes (DPM)) on Day 7.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations conducted on the DPM values were performed with "R" (language and environment for statistical computing and graphics).
Mean values and standard deviations were calculated for the body weights.

Results and discussion

Positive control results:
The positive control substance (hexyl cinnamic aldehyde, 25% in AOO and 10% in AOO) induced positive and a negative reaction, respectively, determined by a DPM/lymph node of 8170.5 and 2908.9, respectively,compared with 1042.8 DPM/lymph node in the vehicle control group, leading to a SI of 7.84 and 2.79, respectively.
The EC3 value was calculated to be 10.6% (w/v) (using the results of 10 and 25% hexyl cinnamic aldehyde).

The historical control data range of the last 10 positive control experiments was 3.7 - 17.6 (= S.I. values)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.82
Test group / Remarks:
5% test group
Key result
Parameter:
SI
Value:
0.74
Test group / Remarks:
10% test group
Key result
Parameter:
SI
Value:
0.54
Test group / Remarks:
20% test group
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
solvent control group (PG)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
No significant lymphoproliferation (SI ≥ 3) was observed for the test item at treatment concentrations of 5, 10 and 20% (w/v) (see "Any other information on results" "Table 2").

DETAILS ON STIMULATION INDEX CALCULATION: SI = DPM/lymph node of a treated group divided by the DPM/lymph node of the respective solvent control group. Before DPM/lymph node values were determined, mean scintillation-background DPM (in main test = 17) was subtracted from test and control raw data. Since the lymph nodes of the animals of a dose group were pooled, DPM/ lymph node was determined by dividing the measured value by the number of pooled lymph nodes (= 8). The following values (DPM/lymph node) were obtained: 1084.8, 893.0, 804.6 and 585.5 in vehicle control (PG), 5, 10 and 20% test groups.

EC3 CALCULATION: Based on the obtained results no EC3 value of the test item could be calculated.

CLINICAL OBSERVATIONS:
No mortality or symptoms of systemic toxicity were observed in any treatment group. No signs of local skin irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS:
Body weights changes were within the range expected for animals of this strain and age.

Any other information on results incl. tables

Table 1: Results of the Pre-screen test

Body weights

Animal No.

Concentration [%]

Body weight (g)

Prior to 1st application

Prior to

Sacrifice (Day 6)

Difference
Day 1 to Day 6

Difference [%]

1

10

21.8

20.8

-1.0

-4.6

2

20

20.9

20.8

-0.1

-0.5

Ear thickness

Animal No.

Concentration [%]

Ear thickness

Prior to

1st application [μm]

Prior to

3rd application [μm]

Prior to

necropsy [μm]

Difference
Day 1 to

Day 3 [µm]

Ear swelling
Day 3 [%]

Difference
Day 1 to

Day 6 [μm]

Ear swelling Day 6 [%]

Mean (Right and Left Ear)

1

10

237.5

245.0

230.0

7.5

3.2

-7.5

-3.2

2

20

245.0

255.0

247.5

10.0

4.1

2.5

1.0

Ear Weights

Animal No.

Concentration [%]

Ear weights after necropsy (mg per animal)

% Increase compared to vehicle values

1

10

25.77

1.5

2

20

26.52

4.4

Mean of historical vehicle controls (propylene glycol): 25.4 mg/animal

Ear Erythema

Animal No.

Score

within

1 h after 1. appl.

24 h
after

1. appl.

within

1 h after 2. appl.

24 h
after

2. appl.

within

1 h after 3. appl.

24 h
after

3. appl.

Day 5

Day 6

1

0*

0

0*

0

0*

0

0

0

2

0*

0

0*

0

0*

0

0

0

Score:

0 = No visible erythema

1 = Very slight erythema

2 = Well defined erythema

3 = Moderate to severe erythema

4 = Severe erythema to formation of eschar which prevents grading of erythema

*substance residuals

Table 2: Stimulation index in mice (main test)

Compound

Concentration [%]

DPM/ lymph node

Stimulation index

Judgement

PG

100

1084.8

1.00

-

Test item

5

893.0

0.82

Negative

10

804.6

0.74

20

585.5

0.54

AOO*

100

1042.8

1.00

-

HCA*

10

2908.9

2.79

Negative

25

8170.5

7.84

Positive

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

HCA = Hexyl cinnamic aldehyde

PG = Polyethylene glycol

- = Not applicable

* = Performed in separate experiment

Table 3: Body weight (main test)

Compound

Concentration [%]

Animal ID No.

Body weight

Day 1 [g]

Day 6 [g]

PG

100

1

19.3

20.2

2

20.3

21.1

3

19.7

19.4

4

20

20.7

Mean ± SD

19.8 ± 0.4

20.4 ± 0.7

Test item

5

5

20

20.4

6

18.3

19.9

7

21.7

22.2

8

22.3

21.4

Mean ± SD

20.6 ± 1.8

21.0 ± 1.0

10

9

18.3

19.3

10

20

20.3

11

19.9

20.4

12

20.6

21

Mean ± SD

19.7 ± 1.0

20.3 ± 0.7

20

13

21.1

20.4

14

19.5

18.8

15

20.4

20.7

16

21.4

22.9

Mean ± SD

20.6 ± 0.8

20.7 ± 1.7

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

HCA = Hexyl cinnamic aldehyde

PG = Polyethylene glycol

SD = Standard deviation

Historical Positive Control values (Feb 2012 - Apr 2016)

Hexyl cinnamic aldehyde (25% in an acetone : olive oil 4:1 (v/v) mixture): SI range = 3.7 - 17.6

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was not a skin sensitizer under the test conditions of the study. 
Executive summary:

The test item formulated in propylene glycol was assessed for its possible skin sensitizing potential according to OECD TG 429. 

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 20%. The highest concentration tested was the highest concentration that could technically be achieved. 

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. 

In this study Stimulation Indices (S.I.) of 0.82, 0.74, and 0.54 were determined with the test item at concentrations of 5, 10, and 20% in PG, respectively. 

The test item was not a skin sensitizer under the test conditions of the study.