trans-1-butyl-4-[trans-4-[(E)-prop-1-enyl]cyclohexyl]cyclohexane

Endpoint summary

Description of key information

Irritation corrosion was studied in vitro in GLP compliant OECD 439 and OECD 492 assays. Both assays were negative.

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:

May 3 - Jul 1, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No. 640/2012 amending, for the purpose of its adaption to technical progress, Regulations (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and the council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Skinethic skin irritation test -42bis Standard operating procedure (SOP) 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Before application, the test item was pre-warmed to 37°C to get a liquid, which was applied neat to the tissues.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

standard model

Vehicle:

unchanged (no vehicle)

Remarks:

No vehicle used in this study; The test item was applied neat to the tissues.

Details on test system:

CELL CULTURE
- Supplier: EpiSkin/SkinEthic Laboratories, Lyon, France)
- Source: human keratinocytes cultured on a polycarbonate filter in conditions which permit their terminal differentiation
- Format: 24 well plate
- Batch: 19-RHE-087
- Expires: May 27, 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF THE TEST MATERIAL AND CONTROL
After the end of the treatment interval, the residual test item was removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer:ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany at 570 nm

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL: 16 µL
NEGATIVE CONTROL: 16 µL (Dulbecco`s Phosphate-Buffered Saline)
POSITIVE CONTROL: 16 µL (5% aqueous solution of sodium dodecyl sulfate in deionised water)

Duration of treatment / exposure:

42 min (± 1 minute)

Duration of post-treatment incubation (if applicable):

42 hours (± 1 hour)

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / Run 1

Value:

95.6

Vehicle controls validity:

not applicable

Remarks:

The test item was applied neat to the tissues

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.663 to 2.041

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.810
Mean viability positive control < 40% 1.3%
SD of group-mean value ≤ 18% 7.7% (positive control)
11.2% (negative control)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.416 1.810
Mean viability positive control ≤ 2.77% 1.3%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 5.0%

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.810	100
Positive Control	42	0.024	1.3
Test Material	42	1.730	95.6

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 439. Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

Objective

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

Study Design

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues.

Results

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the mean tissue viability was 95.6% and, thus, higher than 50%,i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Conclusion

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

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:

Apr 18 - Jun 13, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 9, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT,

Version / remarks:

September 14, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation`s Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Before application, the test item was pre-warmed to 37°C to get a liquid, which was applied neat to the tissues..

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL: 50 µL per tissue

NEGATIVE / VEHICLE CONTROL: 50 µL per tissue

Sterile deionized water was used as negative control.


POSITIVE CONTROL: 50 µL per tissue

Designation: Methyl acetate
Supplier: Merck KGaA
Lot-No.: S7451611
Catalog #: 8.09711
Purity (GC): 99.6%
Appearance: Liquid
Expiration date: June 30, 2022
Storage: 15 to 25°C

Duration of treatment / exposure:

30 ± 2 minutes

Number of animals or in vitro replicates:

in vitro: duplicate design

Irritation parameter:

other: Viability %

Run / experiment:

Run 1 / Experiment 1

Value:

109.2

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:

OTHER EFFECTS:
- Visible damage on test system: No observations

ACCEPTANCE OF RESULTS:
1. The negative control OD is >0.8 and <2.5 (1.607and 1.731).
2. The mean relative viability of the positive control is below 50% of the negative control viability (30.8%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 2.1% to 7.4%) in the same run (for positive and negative control tissues and tissues of single chemicals).

The study met all acceptance criteria

	Mean OD	Mean Viability
Negative Control	1.669	100.0%
Positive Control	0.514	30.8%
Test Item	1.882	109.2%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Executive summary:

Objective

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

Study Design

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcularä-model were treated with the test item, the negative or the positive control for 30 ± 2 minutes. 50 µL of either the test item, the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

Results

After treatment with the negative control (sterile deionized water) the mean OD was 1.669 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 30.8% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 109.2% and, thus, higher than 60%,i.e.according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Conclusion

Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues.

Following treatment with the test item, the mean tissue viability was 95.6% and, thus, higher than 50%,i.e.according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Eye irritation

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcularä-model were treated with the test item, the negative or the positive control for30 ± 2 minutes. 50 µL of either the test item, the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

Following treatment with the test item, the tissue viability was 109.2% and, thus, higher than 60%,i.e.according to OECD 492 the test item is identified as not requiring classification and labeling according to UN GHS (No Category).

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.