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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-12 to 2016-09-22
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-Benzyl-3-carbamoyl-pyridinium, chloride
Cas Number:
5096-13-9
Molecular formula:
C13H13ClN2O
IUPAC Name:
1-Benzyl-3-carbamoyl-pyridinium, chloride
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with UN GHS "Category 2". Depending on the regulatory framework it can also be used to identify non-classified chemicals.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used:
The test was carried out with the reconstituted three-dimensional human skin model EPISKIN-SM™ (SkinEthic). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
- Tissue batch number(s): 16-EKIN-035
- Expiration date: September 05, 2016
- Date of initiation of testing: August 17, 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (exposure duration: 15 min)
- Temperature of post-treatment incubation (if applicable):
a) 37 ± 1 °C (5.0% CO2 for 42 ± 1 h) in 2 ml pre-warmed fresh maintenance medium
b) 37 ± 1 °C (5.0% CO2 for 3 h ± 5 min) in 2 ml pre-warmed MTT medium

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
* washed with DPBS; excess DPBS was removed by blotting bottom with blotting paper;
* after post-incubation: excess medium was removed by blotting bottom on absorbent paper
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: ± 30 nm
- Linear OD range of spectrophotometer: not reported

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
The test substance is considered to be not irritating (the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
PREPARATION AND APPLICATION OF THE TEST ITEM
Firstly, 5 µL distilled water (aqua dest.) was applied to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread on the surface. Afterwards, approximately 10 ± 2 mg (26.3 mg/cm²) of the powder was applied to the epidermis surface. The test item was spread to match size of the tissue using a flat curved spatula. The volume of aqua dest. had to be increased by additional 5 µL to allow spreading of the test item.

NEGATIVE CONTROL: 10 µL DPBS (Gibco, Cat. No. 14040-091, Lot No.: 17371 07).
POSITIVE CONTROL: 10 µL 5% sodium dodecyl sulfate (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277) in aqua dest.
TEST ITEM: 10 ± 2 mg + 10 µL aqua dest.
Duration of treatment / exposure:
15 min +/- 0.5 min
Duration of post-treatment incubation (if applicable):
42 +/- 1 hour
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
main experiment
Value:
99.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 2: Result of the Test Item 1-Benzyl-3-carbamoyl-pyridinium, chloride

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

0.742

0.816

0.676

0.069

0.086

0.086

0.786

0.695

0.766

0.767

0.855

0.689

0.074

0.086

0.081

0.780

0.720

0.793

OD570 (Blank-Corrected)

0.699

0.773

0.634

0.027

0.044

0.044

0.744

0.653

0.724

0.724

0.813

0.647

0.031

0.044

0.039

0.738

0.678

0.751

Mean OD570 of the Duplicates (Blank-Corrected)

0.712

0.793

0.641

0.029

0.044

0.041

0.741

0.666

0.737

Total mean OD570 of 3 Replicate Tissues
(Blank-Corrected)

0.715*

0.038

0.715

SD OD570

0.076

0.008

0.043

Relative Tissue Viability [%]

99.5

110.9

89.6

4.1

6.1

5.8

103.6

93.0

103.1

Mean Relative Tissue Viability [%]

100.0

5.3**

99.9

SD Tissue Viability [%]***

10.6

1.1

5.9

CV [% Viability]

10.6

20.6

5.9

*      Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**   Mean relative tissue viability of the three positive control tissues is ≤ 40%.

*** Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.

Table 3: Test Acceptance Criteria

 

Value

Cut off

pass/fail

Mean OD570 Blank

0.042

< 0.1

pass

Mean Absolute OD570 NK

0.757

0.6NK1.5

pass

Mean Relative Viability PC [%]

5.3

≤ 40%

pass

Max. SD of % Viability

10.6

≤ 18%

pass

 

Table 4: Historical Data

 

OD570 ± 30 nmBlank

Absolute OD570 ± 30 nmNK

Relative Viability PC [%]

Max. Difference Viability [%]

Mean

0.043

0.866

11.7

8.47

SD

0.001

0.120

8.2

8.08

n

67

66

67

286

Historical control data were generated from 2008 - 2015.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed no irritant effects in an in vitro skin irritation study conducted according to OECD 439.
Executive summary:

In an In Vitro Skin Irritation Study according to OECD Guideline 439 (Reconstructed Human Epidermis Test), the potential of the 1-Benzyl-3-carbamoyl-pyridinium, chloride (~ 96 % purity) to induce skin irritation was analysed by using the three-dimensional human skin model EPISKIN-SM™ (SkinEthic) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study the test item was applied topically to the EPISKIN-SM™ tissue for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no irritant effects and is therefore considered to be non-irritating to the skin.