1H-Inden-1-one, 2,3-dihydro-2,6-dimethyl-

Endpoint summary

Description of key information

Skin corrosion in vitro (GLP, OECD TG 431): corrosive (2 independent experiments)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sept-Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: 95.3%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany)

Justification for test system used:

in line with OECD TG 431

Vehicle:

physiological saline

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 0.63cm² size. All tests were performed in triplets for each concentration and each time point (3 min or 60 min).

Adaptation to cell culture conditions:
Inserts with EST-1000 reconstructed human epidermis (0.63 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 corrosivity the EST-1000 inserts were exposed to 50 µL of the test item for 3min (RT) and 60 min in the incubator (3 inserts per period of incubation time), respectively. 0.9 % NaCI (50 µL) treated epidermal models were used as negative controls (determination in triplicates).

Determination of cell viability (MTT):
After the incubation period the inserts were washed carefully in PBS (3 times each insert) and MTT reduction was performed. For viability testing the inserts were placed in new 6 well plates containing 1ml of MTT solution (37°C, 1mg/mL in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 2 hours under cell culture conditions (5% C02, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 300 µL inside and 300 µL outside the insert) on a vertical shaker (at least 60 min). 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 done with "KC4" (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.

Reliability Check
Reliability of the test was confirmed before by interlaboratory validation.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

n=3 samples per time-point for test substance and negative control, respectively

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

50 µl

Duration of treatment / exposure:

60 min

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

65.59

Vehicle controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

5.59

Vehicle controls validity:

valid

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item shows corrosive properties to skin.

Executive summary:

The MTT method has determined following values of viability after 3 min. or after 60 min. of incubation: 65.19% and 5.59%, respectively. Thus, the results show that a corrosive property of the test item was determined by the assay used.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Sept-Nov 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

2004

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

purity: 95.3%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany)

Justification for test system used:

in line with OECD TG 431

Vehicle:

physiological saline

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 0.63cm² size. All tests were performed in triplets for each concentration and each time point (3 min or 60 min).

Adaptation to cell culture conditions:
Inserts with EST-1000 reconstructed human epidermis (0.63 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 corrosivity the EST-1000 inserts were exposed to 50 µL of the test item for 3min (RT) and 60 min in the incubator (3 inserts per period of incubation time), respectively. 0.9 % NaCI (50 µL) treated epidermal models were used as negative controls (determination in triplicates).

Determination of cell viability (MTT):
After the incubation period the inserts were washed carefully in PBS (3 times each insert) and MTT reduction was performed. For viability testing the inserts were placed in new 6 well plates containing 1ml of MTT solution (37°C, 1mg/mL in MTT-assay medium, delivered by Cell Systems®). The tissues were incubated for about 2 hours under cell culture conditions (5% C02, 37°C, max humidity). The extraction of blue formazan was performed in Isopropanol (24 well plates, 300 µL inside and 300 µL outside the insert) on a vertical shaker (at least 60 min). 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 done with "KC4" (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.

Reliability Check
Reliability of the test was confirmed before by interlaboratory validation.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

n=3 samples per time-point for test substance and negative control, respectively

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

50 µl

Duration of treatment / exposure:

60 min

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

42.85

Vehicle controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

7.35

Vehicle controls validity:

valid

Substances are classified as "corrosive (R34)", if the cell viability of the EST 1000 is decreased by more than 50% after 3 min of incubation to the test item, or if the cell viability is less than 15% after 60 min of exposure to the test item. As can be seen from the information in the Table below the test item was detected as positive (exceeding the LD50 value and the 15% viability value, respectively) by the EST-1000 model after an incubation period of 3 min or 60 min.

 

 

 

Compound	Cell viability after 3 min [%]	Cell viability after 60 min [%]	Classification
test item	42.85	7.35	corrosive
negative control	100.00	100.00	negative control

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

The test item shows corrosive properties to skin.

Executive summary:

The MTT method has determined following values of viability after 3 min. or after 60 min. of incubation: 42.85% and 7.35%, respectively. Thus, the results show that a corrosive property of the test item was determined by the assay used.
Because of the fact that both values for viability are near the cut-off this study could be repeated to verify these findings.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results for skin irritation classification Cat. 1 (H314, Causes severe skin burns and eye damage) is warranted according to Regulation (EC) No. 1272/2008 (CLP).

Based on the skin corrosivity testing for eye damage can be waived.