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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:
2011-03-09 - 2011-03-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well-documented GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
OECD Guideline 439 - In Vitro Skin Irritation Reconstructed Human Epidermis Test Method
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Caprolactamdisulfid
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.32 %
- Lot/batch No.: 19779/19777
- Expiration date of the lot/batch: December 14, 2011
- Storage condition of test material: room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: no data
Source strain:
other: not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
Reconstructed tissues
The experiment was carried out on a reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany).
The tissue equivalents were shipped in 24 well cell culture plates on Agarose supplemented with maintenance medium (Kit contents EST-1000; CellSystems, Cat.-No.CS-1001). Inserts were of 0.6 cm2 size. All tests were performed in triplets.

Adaptation to cell culture conditions
Inserts with EST-1000 reconstructed human epidermis (0.6 cm2) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1ml of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1ml new maintenance medium (37°C) for each well.

Environmental conditions
The environmental conditions in the incubator were standardised as follows:
Incubator temperature: 37 ± 2° C
CO2 gas concentration: 5 %
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).

Test item formulation
Test item was used undiluted, i.e. 100% concentration.

Application of the test material and incubation
For testing of chemical induced irritation the EST-1000 inserts were exposed to 30 mg of the test item for 20 min (RT; three inserts per period of incubation time). 0.9% NaCl or 5% SDS (each 30 µl) treated epidermal models were used as negative and positive controls, respectively (determination in triplicates)

Determination of cell viability (MTT)
After the exposure period of 20 minutes the inserts were washed carefully in PBS. After a post – exposure incubation of 42h in the incubator MTT reduction was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µl of MTT solution (37°C, 1 mg/ml in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 2 ml per insert) on a vertical shaker (2 hours). For determination of cell viability the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 96 well format, 200 µl).
Data acquisition and evaluation were done with "Gen5" (software by Bio-Tek).
The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow 3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Sigma, Deisenhofen-Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories and has practically been modified for accurate analysis of cell viability in three dimensional skin models.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg

NEGATIVE CONTROL
Physiological saline solution (0.9 % NaCl, 30µl)

POSITIVE CONTROL
SDS (sodium dodecyl sulfate) solution (5%, 30µl)
Duration of treatment / exposure:
20 min
Number of replicates:
triplicates

Test animals

Species:
other: in vitro test: The experiment was carried out on a reconstructed human epidermis EST-1000
Strain:
other: reconstructed human epidermis EST-1000
Details on test animals and environmental conditions:
not applicable as an in vitro test was performed.

The tissue equivalents were shipped in 24 well cell culture plates on Agarose supplemented with maintenance medium. Inserts were of 0.6 cm² size. All tests were performed in triplets.
Inserts with EST-1000 reconstructed human epidermis (0.6 cm²) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon arrival, 6 well culture plates were pre-filled with 1mL of fresh and cool maintenance medium. The reconstructed tissues were placed into the prepared cell culture plates (1 insert/well) and were adapted to the recommended tissue culture conditions (5% CO2, 37°C, max humidity) afterwards for at least 6 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the medium and replacing it by 1mL new maintenance medium (37°C) for each well.

The environmental conditions in the incubator were standardised as follows:
Incubator temperature: 37 ± 2° C
CO2 gas concentration: 5 %
Humidity: maximum
Occasional deviations from these conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed in a CO2 atmosphere incubator (Heraeus, Osterode-Germany).

Test system

Type of coverage:
open
Preparation of test site:
other: no preparation needed, beside the acclimatisation of the inserts to the tissue culture conditions.
Vehicle:
unchanged (no vehicle)
Controls:
other: no animals were used as this is an in vitro test. Physiological saline solution (0.9 % NaCl, 30 µl) was used as negative control. A SDS (sodium dodecyl sulphate) solution (5%, 30 µl) was used as positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg per insert
- Concentration (if solution): 100 % (undiluted)
- Temperature: room temperature
Duration of treatment / exposure:
20 min
Observation period:
reading was performed immediately after the end of exposure
Number of animals:
not applicable as an in vitro test was performed.
3 inserts were used for each incubation time.
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm² (whole insert)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): washed carefully with phosphate buffered saline
- Time after start of exposure: immediately after end of exposure

SCORING SYSTEM:
For the current test, an irritation potential of a test materials according to UN GHS category 2 is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is less than or equal (≤ ) to 50% of the mean viability of the negative controls.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
101.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none stated

Any other information on results incl. tables

Table 1: Summary of results


Compound

Cell viability [%]

Classification*

Caprolactam disulphide

101.17

Non Irritant (NI)

Positive control

1.34

Irritant (I)

Negative control

100.00

Non irritant (NI)

*: Classification was done in accordance with EU Test Method B.46.

Table 2: Tabular summary of the results

 

Sample No.

Test item

OD mean*

StdDev

% Viability

1-3

Negative control NaCl 0.9%

1.97

0.14

100.00

4-6

Positive control SDS 5%

0.03

0.00

1.34

7-9

Caprolactam disulphide

1.99

0.33

101.17

* 6 values

 Table 3: Classification criteria

In vitro result

In vivo prediction

mean tissue viability < 50

Irritant (I)

mean tissue viability > 50

Non-irritant (NI)

(I):           Irritant, R38 = Skin irritation category 2, H315

(NI):        Non – irritant = no skin irritation category

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The study was performed according to the OECD Guideline 439, EU Method B.46 without deviations and considered to be of the highest quality (reliability Klimisch 1). The vehicle fulfilled validity criteria of the test system. Caprolactam disulphide was shown not to produce any significant cell viability reduction. The test material was considered to be not irritating under the conditions of this test.
Executive summary:

Caprolactam disulphide was subject to an in vitro test to evaluate its irritating properties (Wingenroth, 2011). This study was performed in 2011, according to OECD TG 439 as well as according to EU Test Method B.46, both without deviations. The experiment was carried out on a reconstructed human epidermis EST-1000 for detection of topically applied skin irritants with the test item caprolactam disulphide. This approach was done, applying the sequential testing strategy of OECD testing guideline 404, which allows the use of validated in vitro methods for the determination of skin corrosion /irritation. A 100% concentration was tested on the skin/epidermal equivalents in triplets. For the determination of time related cytotoxic effects the incubation periods were 20 min (30mg per insert plus 30µL 0.9% NaCl to moisten and ensure good contact with the skin; three replicates). After the exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the cell viability was measured to be101.17%in the MTT (Methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, 0.9% NaCl) and positive control (PC, 5% SDS) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to have no skin irritation category, as no significant impact on cell viability after test item exposure was evident.