Registration Dossier

Diss Factsheets

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
1-benzyl-3-carboxylatopyridinium sodium chloride
EC Number:
268-692-9
EC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Cas Number:
68133-60-8
Molecular formula:
C13H12NO2.Cl.Na
IUPAC Name:
1-benzyl-3-carboxylatopyridinium sodium chloride
Details on test material:
- Name of test material (as cited in study report): LUGALVAN BPC dry
- Physical state: solid
- Analytical purity: 95.5 area-%
- Lot/batch No.: 467602
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
other: EpiDerm™ 200 kit
Details on test animals or test system and environmental conditions:
TEST SYSTEM:
- Source: MatTek Corporation, Ashland MA, USA

Three dimensional human epidermis model:
The EpiDermTM model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the Epiderm™ human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

Incubation conditions:
Temperature: ± 37 °C,
CO2: 5 % ,
Humidity: 90-95 %

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume (about 13 mg) of the undiluted test substance
Duration of treatment / exposure:
corrosion test: 3 minutes and 1 hour, respectively
irritation test: 1 hour followed by a 42-hours post-incubation period.
Observation period:
N/A
Number of animals:
N/A
Details on study design:
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently.
If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT.

The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results.

In case that direct MTT reduction occurred, one freeze-killed control tissue per exposure time was treated with, each, the test article and the negative control, in the same way additionally.

Basic procedure
Corrosion test:

From the day of arrival in the laboratory, tissues were kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test-substance application, tissues were transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The pre-incubation medium was replaced with fresh medium immediately before application.

Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) were used.

25 µL de-ionized water was applied first. Thereafter, a bulk volume of 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed with the water.

Control tissues were concurrently applied with 50 µL of de-ionized water (negative control, NC) or with 50 µL of 8 n potassium hydroxide (positive control, PC).

The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment.

Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed. The assay medium was then replaced by MTT solution and tissues were incubated for 3 hours.

After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Irritation test:

On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the pre-incubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours.

Three tissues were treated with the test substance, the PC and NC, respectively.

25 µL sterile PBS was applied first. Thereafter, a bulk volume of 25 µL of the solid test material was applied with a sharp spoon and homogeneously distributed together with the fluid.

Control tissues were concurrently applied with 30 µL of sterile PBS (negative control, NC) or with 30 µL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards.

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.
The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab.

Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours.

After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation.

The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Data evaluation
Table(s) and/or figure(s) of measured parameters presented in the report were produced using PC based tabular calculation software. The mean and individual data were not always rounded but the significant digits were produced by changing the display format. As a consequence, calculation of mean values using the individual data presented in the report will, in some instances, yield minor variations in value.

Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is corrosive or irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues (corrosion test) or three tissues (irritation test) treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA

Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Assay acceptance criterion for the positive control (PC)
Corrosion test: Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%.
Irritation test: 5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, 2 tissues (corrosion test) or 3 tissues (irritation test) are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3 (corrosion test) or if the SD of %-viability is ≤ 20 (irritation test).

EVALUATION OF RESULTS
Corrosive potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with de-ionized water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 15%.
Mean tissue viability
(% of negative control) Prediction
3 min: < 50 corrosive
3 min: ≥ 50 and 1 hour: < 15 corrosive
3 min: ≥ 50 and 1 hour: ≥ 15 non-corrosive

Although the method is not finally validated for categorizing the severity of corrosivity according to certain classification and labeling systems, it is suggested to use the most stringent category for test substances leading to viabilities below 50% after 3 min treatment.

Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Mean tissue viability
(% of negative control) Prediction
≤ 50 irritant
> 50 non-irritant

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: viability [% of control]
Basis:
mean
Score:
89
Remarks on result:
other: corrosion, exposure: 3 min, mean OD570: 2.351
Irritation parameter:
other: viability [% of control]
Basis:
mean
Score:
100
Remarks on result:
other: corrosion, exposure: 1 hour, mean OD570: 2.367
Irritation parameter:
other: viability [% of control]
Basis:
mean
Score:
86
Remarks on result:
other: irritation, mean OD570: 2.030
Irritant / corrosive response data:
Based on the observed results and applying the evaluation criteria it was concluded, that LUGALVAN BPC dry does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU

Categories Display