1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-03-30 - 2011-03-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well-documented GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Principles of method if other than guideline:

The HCE model is currently involved in the eye irritation validation conducted by the COLIPA following ECVAM guidelines. This study is expected to end late this year and results published early 2012. The HCE is produced and commercialized by SkinEthic since 2000 - more than 11 years -, and is the only model made from human corneal cells. The model is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004. Furthermore, this model is recognized as the model of choice and scientifically relevant as documented by several publications.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

EC Number:

245-910-0

EC Name:

1,1'-dithiobis[hexahydro-2H-azepin-2-one]

Cas Number:

23847-08-7

Molecular formula:

C12H20N2O2S2

IUPAC Name:

1-[(2-oxoazepan-1-yl)disulfanyl]azepan-2-one

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

Test animals / tissue source

Species:

other: corneal epithelial tissue (mucosa) without a stratum corneum

Strain:

other: SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France

Details on test animals or tissues and environmental conditions:

not applicable as an in vitro test was performed.

The experiment was carried out on reconstituted human ocular epithelia (SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France).
The model used for this study is a corneal epithelial tissue (mucosa) without a stratum corneum. The ultra-structure (tissue morphology and thickness) is similar to the corneal mucosa of the human eye.

The tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium. The scope of supply contains Maintenance Medium for incubation. Inserts were of 0.5 cm² size. All tests were performed in triplets.

HCE inserts (0.5 cm²) were packed under sterile conditions and were shipped on semi solid agar’s medium. Upon receipt each insert was transferred from the packaging plate to 6 well culture plates containing 1 mL of fresh maintenance medium per well. The HCE inserts were incubated for at least 2 hours (5% CO2, 37°C, max humidity). Afterwards a media change was performed and the HCE inserts were continuing adapted overnight to the recommended tissue culture conditions (5% CO2, 37°C, max humidity). 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 1 mL 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).

The viability of the cells in the model must be sufficiently high to be able to accurately discriminate between the positive and negative control substances. Cell viability is measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative control substance or the test item.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable as an in vitro test. Phosphate Buffered Saline (PBS, 30 µl) was used as negative control. 1H-1,2,4-Triazole-3-thiol (30 mg, plus 30 µl PBS to moisten and ensure good contact with the skin) was used as positive control in three replicates.

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
- Concentration (if solution): 100 % (undiluted)

Duration of treatment / exposure:

60 min

Number of animals or in vitro replicates:

not applicable as an in vitro test was performed.
3 inserts per period of incubation time were used.

Details on study design:

For testing of chemically induced eye irritation the HCE inserts were exposed to 30 mg of the test item for 60 min (RT; three inserts per period of incubation time). PBS (30 µL) or 1H-1,2,4-Triazole-3-thiol (30 mg) treated epidermal models were used as negative and positive controls, respectively, in triplicates.

Determination of cell viability (MTT)
After the exposure period of 60 minutes the inserts were washed carefully with PBS. After a post-exposure incubation of 16h in the incubator MTT reduction assay was performed. For viability testing the inserts were placed in new 24 well plates containing 300 µL of MTT solution (37°C, 0.5 mg/mL in Maintenance medium). 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, 1.5 mL per insert) on a vertical shaker (for at least 2 hours). For determination of cell viability per insert 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 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.

REMOVAL OF TEST SUBSTANCE
- Washing (if done):
- Time after start of exposure:

SCORING SYSTEM:
Cytotoxicity indices (viability decrease measured by MTT)
A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % cell viability

Run / experiment:

mean

Value:

38.32

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

No other effects were reported.

Any other information on results incl. tables

Table 1: Summary of results

Compound	Cell viability [%]	Evaluation
Test item	38.32	irritant
Positive control	3.80	irritant
Negative control	100.00	non-irritant

Table 2: Tabular summary of the results 

Sample No.	Test item	OD mean*	StdDev	% Viability
1-3	Negative control  PBS	0.81	0.01	100.00
4-6	Positive control 1H-1,2,4-Triazole-3-thiol	0.03	0.02	3.80
7-9	Test item	0.31	0.03	38.32

* 6 values

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

The study was performed according to COLIPA following ECVAM guidelines and considered to be of the highest quality (reliability Klimisch 1). The negative and positive control fulfilled validity criteria of the test system. Caprolactam disulphide was shown to produce any significant cell viability reduction. The test material was considered to be irritating to eyes under the conditions of this test.

Executive summary:

Caprolactam disulphide was subject to an in vitro test to evaluate its ocular irritation properties to the eyes after topical exposure of the test item (Wingenroth, 2011). This study was performed in 2011, according to Colipa following ECVAM guidelines. The experiment was carried out on a human epithelial cornea model for detection of eye irritants with the test item caprolactam disulphide. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

The model used is standardised and commercially available (SkinEthic^TMHuman Corneal Epithelial Model (HCE)). A 100% concentration was tested in triplets. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%. For the determination of time related cytotoxic effects the incubation periods were 60 min (30mg per insert plus 30µL 0.9% NaCl to moisten and ensure good contact; three replicates). After the exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was measured to be 38.32% in the MTT (Methylthiazoletetrazolium) conversion assay. The results of the concurrent negative control (NC, PBS) and positive control (PC, 1H-1,2,4 -Triazole-3 -thiol) demonstrated the viability (NC) and sensitivity (PC) of the test model.

Thus, the results show that caprolactam disulphide is considered to be irritating to eyes under the conditions of this test method.