4-(1-ethoxyvinyl)-3,3,5,5-tetramethylcyclohexanone

Endpoint summary

Description of key information

Skin Irritation Endpoint:

The in vitro Episkin positive test is supported by the in vivo Moreno test which also indicates moderate irritation. The human data by Kligman is only at 10% and of limited additional information.

Eye Irritation Endpoint:

The in vitro BCOP test is clearly negative and this is supported by the Klecak in vivo study where only very slight irritation was observed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Kephalis
Appearance: Pale yellow liquid
Batch: PE00134258
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 08 July 2016 (expiry date)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-011).
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source
SkinEthic Laboratories, Lyon, France.

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

The test was performed on a total of 3 tissues per test item together with negative and positive controls. Twenty five μl of the undiluted test item was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μl PBS (negative control) and 3 tissues with 25 μl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test item. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-solution (0.3 mg/ml in PBS). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

Amount/concentration applied:

The liquid test item was applied undiluted (25 μl) directly on top of the tissue.

Species:

other: In vitro study

Irritation / corrosion parameter:

% tissue viability

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Kephalis was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Kephalis did not interact with the MTT endpoint.
The mean absorption at 570 nm measured after treatment with Kephalis and controls are presented in APPENDIX 1, Table 2.
The individual OD570 measurements are presented in APPENDIX 2.
Table 3 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with Kephalis compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Kephalis compared to the negative control tissues was 14%. Since the mean relative tissue viability for Kephalis was below 50% it is considered to be irritant.
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range (See APPENDIX 3). The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

It is concluded that this test is valid and that Kephalis is irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Kephalis
Appearance: Pale yellow liquid
Batch: PE00134258
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 08 July 2016 (expiry date)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Irritation parameter:

in vitro irritation score

Value:

1.1

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:

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Kephalis did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment.
Since Kephalis induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Interpretation of results:

GHS criteria not met

Conclusions:

Evaluation of the eye hazard potential of Kephalis using the Bovine Corneal Opacity and Permeability test (BCOP test). This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Kephalis was tested through topical application for 10 minutes.
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 35 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
Kephalis did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.1 after 10 minutes of treatment.
Since Kephalis induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available