Reaction mass of α-3,3-trimethyl-(αS,1R)-cyclohexanemethanol and α-3,3-trimethyl-(αR,1S)-cyclohexanemethanol

Endpoint summary

Description of key information

An in vitro skin irritation study was performed according to Guideline OECD 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model. As the mean viability was < 50 % the results met the criteria for an irritant response.

Therefore, the test material is classified in category 2 (H315) according to CLP Regulation (EC) No 1272/2008 and UN GHS.

An in vitro eye irritation study was performed according to Guideline OECD 437 and in compliance with GLP.

In Vitro Irritancy Score (IVIS) for the test item was 8. Therefore, the ocular corrosive or severe irritant potential of the test item could not be predicted in this assay.

In an in vitro eye irritation study performed according to Guideline OECD 492 and in compliance with GLP, the mean percent tissue viability of the RhCE replicates treated with the test item was 18.66% versus 22.84 % in the positive control (Methyl acetate).

Therefore, the test material is classified in category 2 (H319) according to CLP Regulation (EC) No 1272/2008 and UN GHS.

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:

04-17 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2013

Test system:

human skin model

Remarks:

human reconstructed epidermis (tissues)

Source species:

human

Cell type:

other: human reconstructed epidermis (tissues)

Cell source:

other: SkinEthic Laboratories, Lyon, France

Vehicle:

unchanged (no vehicle)

Details on test system:

Supplier: SkinEthic Laboratories, Lyon, France.
Selection: At receipt, the pH (color of the agar medium) and temperature indicators were checked to ensure the good quality of the tissues before use.
Storage conditions: At receipt, the living EPISKIN™ tissues were kept at room temperature in their packaging until required.
Description: The EPISKIN™ model consists of an airlifted, living, multilayered epidermal tissue construction (surface 0.38 cm2), reconstructed from normal human epidermal keratinocytes for 13 days and produced in polycarbonate inserts in a serum-free and chemically defined medium. The model features a normal ultra-structure and is functionally equivalent to human in vivo epidermis.

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

other: not applicable

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL ± 2 μL of the test item was applied to the epidermis surface.
- Concentration (if solution): undiluted

CONTROLS
- Negative control: 10 μL ± 2 μL of Dulbecco’s Phosphate-Buffered Saline (D-PBS)
- Positive control: 10 μL ± 2 μL of 5 % (w/v) aqueous solution of Sodium Dodecyl Sulfate (SDS)

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.
At the end of the exposure period, tissues were rinsed and incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

Number of animals:

Triplicate tissues for test item, negative and positive controls

Details on study design:

A new 12-well plate was used for each test and control items: one plate for the test item, one plate for the negative control and one plate for the positive control. Each item was applied onto triplicate tissues.

PRE-INCUBATION OF THE TISSUES ON THEIR DAY OF ARRIVAL (DAY 0)
A volume of 2 mL of pre-warmed maintenance medium was added to the first column of 3 wells of 12-well plates (one plate per item). Then, each EPISKIN™ tissue was transferred into the maintenance medium pre-filled wells (three tissues per plate). The plates were incubated at 37 °C, 5 % CO2 in a humidified incubator for at least 24 h (± 2 h).

TREATMENT OF TISSUES (DAY 1)
A volume of 2 mL of pre-warmed maintenance medium was pipetted into the second column of 3 wells of the 12-well plates for each item, respectively. Then, each test or control item was applied on three tissues for an exposure period of 15 minutes. The items were applied topically to the corresponding tissues and gently spread over the epidermis surfaces to ensure uniform covering of the tissues. The tissues were processed (treatment and rinsing) in the same order and at regular time-intervals to ensure each tissue received an equal exposure period.
The exposure of the tissues to the test and control items was performed at room temperature for 15 minutes (± 1 minute).

RINSING OF TISSUES AND INCUBATION FOR 42 HOURS (DAY 1)
At the end of the treatment period, each tissue was removed from the well of the treatment plate, and rinsed with D-PBS to remove any residual test or control items. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well and the plates were incubated at 37 °C, 5 % CO2 in a humidified incubator for 42 h.

MTT VIABILITY ASSAY (DAY 3)
On Day 3, following the 42 hour post-exposure incubation period: MTT test (MTT Loading/Formazan Extraction) was performed and tissues were incubated for 3 h (± 5 minutes) at 37 °C, 5 % CO2 in a humidified incubator.

OPTICAL DENSITY MEASUREMENTS (DAY 6)
On Day 6, at the end of the formazan extraction period: The optical density was measured at 570 nm using a plate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

4

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

3

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Evaluation of the coloration of tissues at the end of the MTT incubation period: All treated tissues appeared white which was considered to be indicative of dead tissues.
Following a 15 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 4 % with a Standard Deviation of 0 % as assessed by the MTT assay.

Table 7.3.1/1: Individual and mean corrected OD values and tissue viabilities for the test item, the negative and positive controls

 

Group	Tissue No.	OD measurements		Mean ODblank	cOD		Mean cOD	Viability (%)
		1^st	2^nd		1^st	2^nd
Negative control	1	0.993	1.027	0.037	0.956	0.990	0.973	101
	2	0.975	0.988		0.938	0.951	0.945	98
	3	1.019	1.028		0.982	0.991	0.987	102
Positive control	1	0.091	0.090	0.037	0.054	0.053	0.054	6
	2	0.136	0.130		0.099	0.093	0.096	10
	3	0.096	0.088		0.059	0.051	0.055	6
Test item	1	0.073	0.076	0.037	0.036	0.039	0.038	4
	2	0.079	0.072		0.042	0.035	0.039	4
	3	0.070	0.069		0.033	0.032	0.033	3

OD = optical density

cOD = blank corrected optical density

 

Table 7.3.1/2: Mean tissue viability and Standard Deviations for the test item, the negative and positive controls

 

Group	cOD		Viability (%)
	Mean	SD	Mean	SD
Negative control	0.968	0.021	100	2
Positive control	0.068	0.024	7	2
Test item	0.036	0.003	4	0

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material is classified in category 2 (H315) according to CLP Regulation (EC) No 1272/2008 and UN GHS.

Executive summary:

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

The test item was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

 

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

 

All treated tissues appeared white which was considered to be indicative of dead tissues. Following a 15 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 4% with a Standard Deviation of 0%. As the mean viability was < 50 % the results met the criteria for an irritant response.

 

Therefore, the test material is classified in category 2 (H315) according to CLP Regulation (EC) No 1272/2008 and UN GHS.

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:

supporting study

Study period:

12-13 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2013

Species:

other: Bovine eye

Details on test animals or tissues and environmental conditions:

Origin: bovine eyes were obtained from EVA, Saint-Pierre-sur-Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: Bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CiToxLAB France: The eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL (± 8 μL) was applied on each cornea
- Concentration (if solution): undiluted

Duration of treatment / exposure:

The test item was evaluated by using a treatment time of 10 minutes ± 30 seconds.

Duration of post- treatment incubation (in vitro):

2 hours ± 10 minutes in a water bath at 32 ± 1°C.

Number of animals or in vitro replicates:

Total: 9 corneas - 3 corneas/group for test item, negative and positive controls

Details on study design:

Dose formulation application:
As the test item was a non-viscous liquid, the closed-chamber method was used as follows: the test and control items were introduced into the anterior chamber of the corneal holder through the dosing holes, to cover the epithelial side of the cornea. Then the dosing holes were sealed.

Treatment of corneas:
Corneas obtained from freshly slaughtered cattle (from abattoir) were mounted in corneal holders with the endothelial side against the O-ring of the posterior chamber. For pre-incubation, both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) and incubated for 1 h and 5 minutes ± 5 minutes at 32 ± 1 °C. Then MEM was removed & then refilled with fresh cMEM and corneas were examined macroscopically for any defects. Then, the opacity of the cornea was measured to obtain OPT0. The medium was removed from anterior chamber and the test item was applied onto the epithelium of the cornea. After application of the dose formulation, the holders were incubated, vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at 32 ± 1 °C, for 10 minutes. At the end of the exposure period, the test item was removed from the anterior chamber and the corneas were rinsed up to six times with pre-warmed cMEM containing phenol red. Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red. Following the 10-minute treatment, the holders were incubated horizontally (corneas placed vertically) for 2 h ± 10 minutes in a water bath at 32 ± 1 °C. After completion of the 2 h incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (32 °C), then the second opacity measurement (OPT2) was performed.

Permeability determination
- Application of sodium fluorescein: Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium of the anterior chamber was removed and the chamber received 1 mL of a validated fluorescein solution (4 mg/mL). For each series of three corneas, a chronometer started from the fluorescein application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at 32 ± 1 °C for 90 ± 5 minutes. After incubation the medium in the posterior chamber of each holder was decanted and retained. Then optical density at 490 nm (OD490) was measured using the spectrophotometer.

CONTROLS:
Negative control: 0.9 % sodium chloride solution
Positive control: 10% sodium hydroxide solution

OTHERS:
- Macroscopic examination: After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium. Then, the corneas were discarded.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

14

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

8

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

3

Negative controls validity:

valid

Positive controls validity:

valid

Irritant / corrosive response data:

- Macroscopic examinations: No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.
- In Vitro Irritancy Score (IVIS) for test item and positive control were 8 and 187, respectively.

Other effects:

None

Table 7.3.2/1: Eye irritation – results

GROUP		OPACITY				PERMEABILITY		SCORE
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490 nm	cOD490 nm
Negative control	22	3	2	-1.0	-	0.002	-	-
	35	3	2	-1.0	-	0.004	-	-
	38	3	2	-1.0	-	0.021	-	-
	Mean	-	-	-1.0	-	0.009	-	-
	SD	-	-	0.0	-	0.010	-	-
Test item	11	2	12	10.0	11.0	0.217	0.208	14
	9	3	8	5.0	6.0	0.110	0.101	8
	41	3	1	-2.0	-1.0	0.289	0.280	3
	Mean	-	-	-	5.3	-	0.196	8
	SD	-	-	-	6.0	-	0.090	5.5
Positive control	42	3	129	126.0	127.0	5.248	5.239	206
	18	3	111	108.0	109.0	5.312	5.303	189
	16	4	115	111.0	112.0	3.736	3.727	168
	Mean	-	-	-	116.0	-	4.756	187
	SD	-	-	-	9.6	-	0.892	18.9

 

OD: optical density

cOD: corrected optical density

cOPT: corrected corneal opacity Positive control: 10% NaOH solution

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after the 2 h recovery period

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this study, the ocular corrosive or severe irritant potential of test item α-3,3-trimethylcyclohexanemethanol multiconstituent could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage.

Executive summary:

In an in vitro eye irritation study performed according to OECD Guideline 437 and in compliance with GLP, 750 μL (± 8 μL) of undiluted test item, α-3,3 -trimethylcyclohexanemethanol multiconstituent was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 2 h at 32°C. Three corneas were used for each treated series (undiluted test item; negative control; positive control). Before the treatment, a first opacity measurement was performed using an opacitometer. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control. In Vitro Irritancy Score (IVIS) for test item and positive control were 8 and 187, respectively. 

Therefore, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An in vitro skin irritation study was performed according to OECD Guideline 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model.

The test item was applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 h at 37°C, 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In the preliminary tests, the test item was found not to have direct MTT reducing properties or coloring potential.

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid.

All treated tissues appeared white which was considered to be indicative of dead tissues. Following a 15 minutes exposure and 42 h of recovery period, the relative mean viability of the tissues treated with the test item was 4% with a Standard Deviation of 0%. As the mean viability was < 50 % the results met the criteria for an irritant response.

Therefore, the test material is classified in category 2 (H315) according to CLP Regulation (EC) No. 1272/2008.

In an in vitro eye irritation study performed according to OECD Guideline 437 and in compliance with GLP, 750 μL (± 8 μL) of undiluted test item,α-3,3 -trimethylcyclohexanemethanol multiconstituentwas applied to isolated bovine corneas for 10 minutes followed by an incubation period of2 h at 32°C. Three corneas were used for each treated series (undiluted test item; negative control; positive control). Before the treatment, a first opacity measurement was performed using an opacitometer. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate anIn VitroIrritancy Score (IVIS).

No notable opaque spots or irregularities were observed on negative control corneas. Fluorescein fixation was observed on all test item-treated corneas. Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.In VitroIrritancy Score (IVIS) for test item and positive control were 8 and 187, respectively. 

Therefore, the ocular corrosive or severe irritant potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage.

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to Guideline OECD 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test substance was applied, as supplied, at the dose of 50 µL to two DPBS pre-treated RhCE (EpiOcular™ tissue model) during 30 minutes at 37°C, 5% CO2, 95% humidity (standard culture conditions). The exposure period was followed by extensive rinsing with DPBS at room temperature, a 12-minute post-exposure immersion period at room temperature and a 2-hour and 2-minute post-exposure incubation at standard culture conditions. The tissue viability was measured by performing a MTT assay.

In the preliminary tests, the test substance was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent tissue viability of the RhCE replicates treated with the test substance was 18.66%, versus 22.84% in the positive control (Methyl acetate).

Therefore, the test substance was identified as potentially requiring classification and labelling according to UN GHS and CLP Regulation (Category 2 or Category 1). The results of a corrosivity study (OECD 437) have to be taken into account to confirm the classification of the substance.

Justification for classification or non-classification

An in vitro skin irritation study was performed according to Guideline OECD 439 and in compliance with GLP, using the EPISKIN^TM reconstructed human epidermis model. As the mean viability was < 50% the results met the criteria for an irritant response.

Therefore, the test material is classified in category 2 (H315) according to CLP Regulation (EC) No. 1272/2008 and UN GHS.

An in vitro eye irritation study was performed according to Guideline OECD 437 and in compliance with GLP.

As the In Vitro Irritancy Score (IVIS) for the test item was 8, the ocular corrosive or severe irritant potential of the test item could not be predicted in this assay.

In an in vitro eye irritation study performed according to OECD Guideline 492 and in compliance with GLP, the mean percent tissue viability of the RhCE replicates treated with the test item was 18.66% versus 22.84 % in the positive control (Methyl acetate).

Therefore, the test material is classified in category 2 (H319) according to CLP Regulation (EC) No 1272/2008 and UN GHS.