Cyclohexanone, 4-methoxy-

Endpoint summary

Description of key information

Skin irritation in vivo (Rabbit, GLP, OECD TG 404): not skin irritating

Skin corrosion in vitro (RhE, GLP, OECD TG 431): not skin corrosive

Eye irritation (Rabbit, GLP, OECD TG 405): irritating to eye

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:

supporting study

Study period:

May - June 2006

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

GLP compliance:

yes (incl. QA statement)

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

88.46

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

72.01

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

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.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was not characterised by a significant impact on cell viability after the 3 min and after the 60 min period.
Thus, it should not be labelled as corrosive to skin.

Executive summary:

This study for predicting non-specific, corrosive potentials of compounds by using reconstructed human skin (RHS) was performed in 2006. The experiment was carried out on a reconstructed human epidermis EST-1000 (CellSystems, St. Katharinen, Germany) for detection of topically applied skin corrosives with the test item.

A 100% concentration was tested on the skin/epidermal equivalents in triplicate. For the determination of time related cytotoxic effects the incubation periods were 3 min and 60 min, respectively. Thus the study was conducted in accordance with the OECD 431 guideline draft as well as with an EC draft guideline (amending Council Directive 67/548/EEC, B.40 Skin corrosion) using the test item concentrations and incubation periods recommended there. 

The test item was applied at a 100% concentration, i.e. 50 pi per insert. The MTT (Methylthiazoletetrazolium) method has determined the following values of viability after 3 min. or after 60 min. of incubation: 88.46 % and 72.01%, respectively.
Thus, the results show that no corrosive property of the test item was determined by the assay used.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

1967

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

no

Duration of treatment / exposure:

4 hours

Observation period:

7 days

Number of animals:

3 animals

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48/72 h

Score:

0.77

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Interpretation of results:

GHS criteria not met

Conclusions:

According to classification criteria the test item is not an irritant to the skin.
There were no systemic intolerance reactions.

Executive summary:

The irritant / corrosive potential of the test item was studied on the skin of rabbits. The method used complied with the OECD - Guideline for Testing of Chemicals No. 404 -" Acute Dermal Irritation/Corrosion"; adopted: 24th April 2002; EEC Directive 67/548 Annex V - Method B.4. (1967) in its current version, and the principles of the EPA Health Effects Test Guidelines (OPPTS 870.2500), United States, EPA 712-C-98-196 (1998).

According to classification criteria 4-Methoxycyclohexanone is not an irritant to the skin.
There were no systemic intolerance reactions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - July 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

1967

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Vehicle:

unchanged (no vehicle)

Controls:

no

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3 animals

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

1.9

Max. score:

4

Reversibility:

fully reversible within: 14 days

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

2.76

Max. score:

3

Reversibility:

fully reversible within: 14 days

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

1.53

Max. score:

4

Reversibility:

fully reversible within: 14 days

Interpretation of results:

irritating

Conclusions:

According to classification criteria the test item is irritating to eyes with full reversibility within 14 days.
There were no systemic intolerance reactions.

Executive summary:

The irritant / corrosive effects of the test item were tested on the eye of rabbits. The method used complied with the OECD - Guideline for Testing of Chemicals No. 405 -"Acute Eye Irritation/Corrosion"; adopted: 24th April 2002; EEC Directive 67/548 Annex V - Method B.5. (1967) in its current version, and the principles of the EPA Health Effects Test Guidelines (OPPTS 870.2400), United States, EPA 712-C-98-195 (1998), with the exception that recognized differences exist between OECD - Guideline 405 and EPA - Guideline OPPTS 870.2400.

 

According to classification criteria the test item is irritating to eyes with full reversibility within 14 days.
There were no systemic intolerance reactions.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the study results for skin irritation no classification is warranted and for eye irritation allocation to category 2 (H319) according to Regulation (EC) No. 1272/2008 (CLP), ANNEX I, is warranted.