ethyl 2-acetyl-4-methyltridec-2-enoate

Endpoint summary

Description of key information

In vitro skin irritation study:

This report describes the ability of GR-86-6599 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of GR-86-6599 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-86-6599 compared to the negative control tissues was 99%. Since the mean relative tissue viability for GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.

In vitro eye irritation study:

Evaluation of the eye hazard potential of GR-86-6599 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 GR-86-6599 was tested through topical application for 10 minutes. The study procedures described in this report were based on the most recent OECD guideline.

GR-86-6599 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment.

Since GR-86-6599 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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

Study period:

22 November 2016 to 27 Feruary 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Remarks:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2 , Batch no.: 17-EKIN-008, See APPENDIX 4).

Source species:

human

Cell type:

non-transformed keratinocytes

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:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-047).
This model is a three-dimensional human epidermis model, which consists of adult humanderived 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.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

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

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

The test was performed on a total of 3 tissues per test item together with negative and positive controls

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with GR-86-6599 compared to the negative control tissues was 99%. Since the mean relative tissue viability for GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.
The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

Since the mean relative tissue viability for GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.
Finally, it is concluded that this test is valid and that GR-86-6599 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report and should not be classified according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

In vitro skin irritation test with GR-86-6599 using a human skin model.

This report describes the ability of GR-86-6599 to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)). The possible skin irritation potential of GR-86-6599 was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 43 of GR-86-6599 was a yellow liquid. GR-86-6599 was applied undiluted (25 μl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as

the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

GR-86-6599 did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The nonspecific

reduction of MTT (NSMTT) by GR-86-6599 was -3.1% of the negative control tissues. Since the %NSMTT was ≤ 0.0, there was no correction applied on the ODs of the test item treated viable 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 GR-86-6599 compared to the negative control tissues was 99%. Since the mean relative tissue viability for

GR-86-6599 was above 50% after 15 ± 0.5 minutes treatment GR-86-6599 is considered to be non-irritant.

The positive control had a mean cell viability of 22% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage

viability of three tissues treated identically was less than 6%, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that GR-86-6599 is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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

Study period:

22 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

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)

Version / remarks:

2013

Deviations:

yes

Remarks:

One of the negative control eyes was excluded since the final opacity value of 4.2 was slightly outside the historical data range. Since the other two eyes met the criteria and the test item results were not influenced, no affect on the study outcome.

GLP compliance:

yes

Species:

cattle

Details on test animals or tissues and environmental conditions:

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.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied as it is (750 μl) directly on top of the corneas

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 10+/- 1 minutes at 32 +/- 1°C

Duration of post- treatment incubation (in vitro):

Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1°C.

Number of animals or in vitro replicates:

Triplicate for each group (test item, negative and positive control)

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes of treatment

Value:

-0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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 45 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.
GR-86-6599 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment. Since GR-86-6599 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Interpretation of results:

GHS criteria not met

Conclusions:

GR-86-6599 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment. Since GR-86-6599 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Executive summary:

Evaluation of the eye hazard potential of GR-86-6599 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 GR-86-6599 was tested through topical application for

10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 43 of GR-86-6599 was a yellow liquid with a purity of 96.5%. The test item was applied as it is (750 μl) directly on top of the corneas.

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 45 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.

GR-86-6599 did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -0.2 after 10 minutes of treatment.

Since GR-86-6599 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

Justification for classification or non-classification

Based on the in vitro skin and eye irritation studies, Scentaurus Clean (GR-86-6599) does not have to be classified and has no obligatory labelling requirement for skin (result > 50%) and eye (IVIS <= 3) irritations according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).