Disodium adipate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012+

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

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. Study conducted according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Principles of method if other than guideline:

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 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.

Methods
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 (4). Furthermore, this model is recognized as the model of choice and scientifically relevant as documented by several publications (5,6,7).
These tests are also related to OECD 405, Attachment March 2000.

Reconstructed tissues
The experiment was carried out on reconstituted human ocular epithelia
(SkinEthicTM Human Corneal Epithelial Model (HCE); SkinEthic, France).

The tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium. The scope of supply contains Maitenance Medium for incubation (SkinEthic, Cat.-No.RHC/S/5). Inserts were of 0.5 cm2 size. All tests were performed in triplets.

Adaptation to cell culture conditions
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 1ml 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 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 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.
(*plus 30 µl PBS to moisten and ensure good contact with the skin)

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 (Sigma, Deisenhofen-Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results in various laboratories (2) and has practically been modified for accurate analysis of cell viability in three dimensional skin models.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium adipate

EC Number:

231-293-5

EC Name:

Disodium adipate

Cas Number:

7486-38-6

Molecular formula:

C6H10O4.2Na

IUPAC Name:

hexanedioic acid

Test material form:

other: solid

Details on test material:

Content: 97-100%

Test animals / tissue source

Species:

other: human corneal epithelial cell (HCE) construct

Strain:

other: human corneal epithelial cell (HCE) construct

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Duration of treatment / exposure:

After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 72% (rounded) as measured by a MTT conversion assay.

Observation period (in vivo):

After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 72% (rounded) as measured by a MTT conversion assay.

Number of animals or in vitro replicates:

not applicable

Results and discussion

In vivo

Results

Irritation parameter:

other: cell viability

Basis:

mean

Time point:

other: 60 min

Score:

72

Remarks on result:

other: viability in %

Any other information on results incl. tables

After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 72% (rounded) as measured by a MTT conversion assay.A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50%.

Applicant's summary and conclusion

Conclusions:

Disodium adipate is identified as non-irritant under the conditions of this in vitro assay.

Executive summary:

Disodium adipate was not characterised by a significant impact on cell viability after test item exposure.

 

Thus, Disodium adipate is identified as non-irritant under the conditions of this in vitro assay.