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Description of key information

Skin Sensitisation: Negative for skin sensitisation; OECD 442B. W. Chung., 2018

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 May - 26 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted under GLP in accordance with the international guideline.
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
July 2010
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA
Remarks:
CBA/J, SPF
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ORIENTBIO INC.,Republic of Korea
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 19.0 - 24.9 g
- Fasting period before study: N/A
- Housing: Animals were individually housed in Polysulfone cage 200Wx320Dx140H (mm), 2–5 animals/cage (during the quarantine-acclimation period) / 2–3 animals/cage (during the study)
- Diet (e.g. ad libitum): Pelleted rodent chow; Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (lot no. 2918C-012918MA) placed in feeders and provided ad libitum. 

- Water (e.g. ad libitum): Public tap water in Cheongju-si was filtered and irradiated by ultraviolet light and provided ad libitum in a quarantine room and by an automatic watering system in an animal room. 

-Acclimation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1−22.0°C
- Humidity (%): 47.5−67.2%
- Air changes (per hr): 10−15 clean, fresh, filtered air changes per hour
- Photoperiod (hrs dark / hrs light): 12 : 12
IN-LIFE DATES: 11 May - 04 June 2018
Vehicle:
dimethylformamide
Concentration:
10% , 25%, 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: 50% in DMF
- Irritation: Yes 
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU- ELISA
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA (BrdU-ELISA) if the following criteria are fulfilled:
The stimulation index (SI), was calculated to be >1.6 in all groups.

TREATMENT PREPARATION AND ADMINISTRATION:
The required amount of the test substance was weighed on an electronic balance (CPA224S, LA230S, CP323S, ENTRIS423i-1S, Sartorius, Germany) and placed in a bottle. A small amount of vehicle, DMF, was added and mixed using a vortex mixer until suspended. The vehicle was gradually added to yield the desired concentration. All preparations were conducted just prior to use. 

A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. The dose level of the positive substance was selected at 25%, which is the dose recommended in the guideline. Negative control animals were dosed with the vehicle.
The dose range finding part of this study was conducted under Non-GLP conditions. . The test substance was suspended in DMF at a preliminary solubility test. Therefore, dose levels were selected from a series of appropriate concentrations such as 50, 25, 10, 5 and 2.5%. 

Based on the result of the dose range finding study, the high dose did not show toxicity, thus the high dose level for the main study was selected at 50% with two additional lower dose levels of 25 and 10%. In addition, the positive (25% HCA) and negative control (Vehicle) groups were included in the main study.

Ear thickness measurement (one time) was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 (the day of necropsy).
The required amount of the BrdU was weighed on an electronic balance (CPA224S, Sartorius, Germany) and placed in a bottle. A small amount of phosphate buffered saline (PBS, Lot No.: 0000673033, Lonza, U.S.A.) was added and mixed using a vortex mixer until dissolved. The PBS was gradually added to yield the desired concentration. 
A volume of 0.5 mL (5 mg/mouse) of BrdU (Lot No.: HMBG1077V, Sigma- Aldrich Co., U.S.A.) (10 mg/mL) solution was injected inter-peritoneally on Day 5. 

Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. Using a punch for skin biopsy of 6 mm in diameter, centering around both ears and avoiding the flexed part of the inner ear, the inner tissue of the ear was removed about 1 mm from the outside of the ear and tissues of both sides were weighed together. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS, Lot No.: 0000673033, Lonza, U.S.A.) for each animal. For each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by 70 μm nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume (25 mL) was based on the mean absorbance within 0.1–0.2 in the NC group. 

BrdU was measured by ELISA (PowerWave XS, BioTek Instruments, Inc., U.S.A.) using a commercial kit (Lot No.: 29134900, Roche Diagnostic GmbH, Mannhein, Germany). Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After dispensing, centrifugation (300 g, 10 min) was allowed the cells to settle to the bottom and remove the PBS. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added and allowed to produce chromogen. Absorbance at 370 nm (Emission wavelength, em) with a reference wavelength of 492 nm (Reference wavelength, ref) was measured.
Statistics:
Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index. Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data.Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons.
Positive control results:
The animals treated with the positive control item alpha-Hexylcinnamaldehyde (25% (w/v)) showed a distinctly positive response (mean S.I. of 3.31), corroborating the validity of the assay.
Key result
Parameter:
SI
Value:
0.82
Variability:
Yes
Test group / Remarks:
G2 - 10%
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
0.82
Variability:
Yes
Test group / Remarks:
G3 - 25%
Remarks on result:
other: Negative
Key result
Parameter:
SI
Value:
0.72
Variability:
Yes
Test group / Remarks:
G4 - 50%
Remarks on result:
other: Negative
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: BrdU was measured by ELISA at 10, 25 and 50% the mean stimulation indices of 0.82, 0.80 and 0.72 were observed, respectively.

DETAILS ON STIMULATION INDEX CALCULATION: BrdU labelling index/mouse of test substance group
/ Mean of BrdU labelling index of negative control

EC3 CALCULATION: Not determiened.

CLINICAL OBSERVATIONS: There were no abnormal clinical signs or deaths in any dosing group during the observation period. There were no significant differences in the mean erythema score or mean ear thickness at all dose groups.


BODY WEIGHTS: Within normal limits

Table 2.       Mean Stimulation Index results

Group/ Dose (%)

BrdU labelling index

Stimulation index

G1. 0

0.23

1.00

G2. 10

0.19

0.82

G3.  25

0.18

0.80

G4.  50

0.16

0.72

G5. 25

0.75

3.31# 

G1: N,N-dimethylformamide, G2-G4: Calcium manganese titanium oxide, G5: α-hexylcinnamaldehyde

# p<0.05, Significant difference from the negative control group (G1) by Steel's t-test.

Table 3. Mean Ear Thickness

Group/ Dose (%)

Ear thickness (mm)

Time after administration (days)

1

3

6

G1. 0

0.19

0.20

0.21

G2.  10

0.19

0.20

0.21

G3.  25

0.19

0.20

0.21

G4.  50

0.19

0.20

0.21

G5. 25

0.19

0.21

0.23

Table 4. Mean Erythema scores

Group/ Dose (%)

Erythema scores

Time after administration (days)

1

2

3

4

5

6

G1. 0

0

0

0

0

0

0

G2. 10

0

0

0

0

0

0

G3. 25

0

0

0

0

0

0

G4. 50

0

0

0

0

0

0

G5. 25

0

1 

#

1 

# 

#

1 

# 

#

2 

# 

#

2 

# 

#

# p<0.05, Significant difference from the negative control group (G1) by Steel's t-test.

## p<0.01, Significant difference from the negative control group (G1) by Steel's t-test.

Table 5. Mean Body weight

Group/ Dose (%)

Body Weight (g)

Time after administration (days)

1

6

G1. 0

22.3

22.2

G2. 10

22.4

22.5

G3. 25

22.4

22.2

G4. 50

22.7

22.6

G5. 25

22.5

22.0

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test. Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

OECD 442B (2018) - In a dermal sensitisation study with test item in young female adult mice (CBA/J mice) were tested using the Local Lymph Node Assay: BrdU- ELISA (LLNA: BrdU- ELISA). 

Based on the result of the dose range finding study, the high dose did not show toxicity, thus the high dose level for the main study was selected at 50% with two additional lower dose levels of 25 and 10%.  In addition, the positive (25% HCA) and negative control (Vehicle) groups were included in the main study.

As a result of the study, no abnormal clinical signs or deaths were observed in any test substance group during the observation period.  In the test substance groups, the body weight, erythema score, ear thickness, ear weight and stimulation index (SI) were not significantly different when compared to the negative control group. The stimulation index (SI), which is an index of skin sensitisation, was calculated to be <1.6 in all groups.

In the positive control group at 25%, the erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group, and the stimulation index (SI) was calculated to be 3.31, which is over 1.6.

Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

OECD 442B (2018) - In a dermal sensitisation study with test item in young female adult mice (CBA/J mice) were tested using the Local Lymph Node Assay: BrdU- ELISA (LLNA: BrdU- ELISA). 

Based on the result of the dose range finding study, the high dose did not show toxicity, thus the high dose level for the main study was selected at 50% with two additional lower dose levels of 25 and 10%.  In addition, the positive (25% HCA) and negative control (Vehicle) groups were included in the main study.

As a result of the study, no abnormal clinical signs or deaths were observed in any test substance group during the observation period.  In the test substance groups, the body weight, erythema score, ear thickness, ear weight and stimulation index (SI) were not significantly different when compared to the negative control group. The stimulation index (SI), which is an index of skin sensitisation, was calculated to be <1.6 in all groups.

In the positive control group at 25%, the erythema score, ear thickness, ear weight and stimulation index were significantly increased when compared to the negative control group, and the stimulation index (SI) was calculated to be 3.31, which is over 1.6.

Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System or the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Justification for classification or non-classification

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.