[trans(trans)]- 4-Methyl-4'-propyl-1,1'-bicyclohexyl

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.

In addition skin irritation was studied in vivo according to OECD 404 to support registration in China. In this assay the dermal exposure to the test substances resulted in severe skin effects which did not resolve

within the 14 day observation period. Therefore, the test item is considered to be a skin irritant. An in vivo eye irritation study was thus not performed due to animal welfare reasons.

According to the Regulation (EC) No 1272/2008, a skin irritant substance may be considered as leading to eye irritation. As worst case approach, the test item was therefore considered to be irritating to eye despite the negative result from the in vitro eye irritation study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

December 5, 2017 - May 4, 2018

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: 18-RHE-037
- Expires: April 16, 2018

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:

55.9

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 1.507 to 1.756

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.2 1.660
Mean viability positive control < 40% 1.3%
SD of group-mean value ≤ 18% 15.4% (positive control)
8.1% (negative control)

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

Acceptance Criterion Result
Mean OD negative control ≥ 1.455 1.660
Mean viability positive control ≤ 2.97% 1.3%

Test Item Data Acceptance Criteria:

Acceptance Criterion Result
SD of group-mean value ≤ 18% 8.4%

The study met all acceptance criteria.

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	42	1.660	100
Positive Control	42	0.022	1.3
Test Material	42	0.928	55.9

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 tissue viability was 55.9% 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).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

01 Feb 2019 - 25 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River France, L’Arbresle, France
Number of Animals: 1 Female.
Age at the Initiation of Dosing: A young adult animal (approximately 27 weeks old) was selected.
Weight at the Initiation of Dosing: 4210 g.
Acclimatisation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C
- Humidity (%): 57 to 58%.
- Air changes (per hr): 10 or greater
- Photoperiod (hrs dark / hrs light): 12/12 hours

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

4 h

Observation period:

14 days

Number of animals:

1 female

Details on study design:

TEST SITE
Approximately 24 hours before dosing, the dorsal fur was clipped with an electric clipper, exposing an area of approximately 150 square centimeters (10x15 cm). To facilitate scoring,
the treated skin area was re-clipped at least 3 hours before the observations.
The animal was treated by dermal application of 0.5 grams of the test item. The test item was applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleaned of residual test item using tap water.

SCORING SYSTEM:
- Method of calculation:

The skin reactions were assessed at approximately 1, 24, 48 and 72 hours and 7 and 14 days after the removal of the dressings and test item. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of the animal served as control.
The irritation was assessed according to the following numerical scoring system. At each observation, the highest scores given were recorded:

Erythema and eschar formation:
No erythema .............................................................................................................................................................. 0
Very slight erythema (barely perceptible) ................................................................................................................. 1
Well-defined erythema ............................................................................................................................................... 2
Moderate to severe erythema..................................................................................................................................... 3
Severe erythema (beef redness) * ............................................................................................................................ 4
*. Where signs of necrosis or corrosion (injuries in depth) prevent erythema scoring, the maximum grade for erythema (= 4) is given.

Oedema formation:
No oedema.................................................................................................................................................................. 0
Very slight oedema (barely perceptible)..................................................................................................................... 1
Slight oedema (edges of area well-defined by definite raising)................................................................................. 2
Moderate oedema (raised approximately 1 millimeter)................................................................................................ 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) ................................. 4

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3.3

Max. score:

4

Reversibility:

not fully reversible within: 14 days

Irritant / corrosive response data:

Four hours exposure to 0.5 g of the test item resulted in severe erythema and severe oedema in the treated skin area of the rabbit. Gray necrosis of the edges of the application area were noted from 24 hours after application until 72 hours after application. Furthermore, reduced flexibility of the skin, scabs, scaliness, superficial fissures and bald skin were noted during the observation period.
The skin irritation did not completely resolve within the 14 days observation period.
Based on these severe irritation results, no further animals were treated.

Other effects:

No signs of systemic toxicity were observed in the animal during the test period and no mortality occurred.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of the test material to the intact rabbit skin. However, the test item is considered to be irritating to skin.

Executive summary:

The objective of this primary skin irritation study was to assess the possible irritation or corrosion potential of a single dose of the test material when administered to the intact skin of rabbits.

The study was carried out in compliance with the guidelines described in:

• OECD No. 404 (2015) "Acute Dermal Irritation / Corrosion".

• EC No 440/2008, part B: "Acute Toxicity: Dermal Irritation/Corrosion".

• EPA, OPPTS 870.2500 (1998), "Acute Dermal Irritation".

• JMAFF Guidelines (2000), including the most recent revisions.

One rabbit was exposed to 0.5 grams of the test material, by application onto clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours and 7 and 14 days after exposure.

Exposure to the test material resulted in severe skin effects which did not resolve within the 14 day observation period. Severe erythema and severe oedema were seen in the treated skin area of the rabbit.

Gray discoloration (signs of necrosis) at the edges of the application area were noted from 24 hours after application until 72 hours after application.

Furthermore, reduced flexibility of the skin, scabs, scaliness, superficial fissures and bald skin were noted during the observation period. The skin irritation did not completely resolve within the 14 days observation period.

No evidence of full thickness destruction of the skin or scar tissue was observed during the observation period, indicating that no corrosion of the skin had occurred by dermal application of the test material to the intact rabbit skin.

Based on these results, the test item is considered to be irritating to skin.

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

Study period:

January 29, 2018 - June 19, 2018

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: MatTek In Vitro Life Science Laboratories
Lot-No.: 022118ISA
Catalog #: TC-MA
Purity (GC): 99.7%
Appearance: Colorless liquid
Expiration date: February 21, 2019
Storage: 15 to 30°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:

100.5

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 (2.199 and 2.202).
2. The mean relative viability of the positive control is below 50% of the negative control viability (48.9%).
3. The difference of viability between the two relating tissues of a single chemical is <20% (values between 0.1% to 7.2%) 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	2.201	100.0%
Positive Control	1.077	48.9%
Test Item	2.211	100.5%

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 (EpiOcularä) 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.

Results

After treatment with the negative control (sterile deionized water) the mean OD was 2.201 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 48.9% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 100.5% 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:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments), the test substance should be classified as: skin and eye irritant (Category 2).