Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Endpoint summary

Description of key information

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

In an in vitro eye irritation study performed according to OECD Guideline 437 and in compliance with GLP, In Vitro Irritancy Score (IVIS) for test item and positive control were 20 and 187, respectively. 

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 4.88% versus 22.84 % in the positive control (Methyl acetate).

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:

other: human reconstructed epidermis (tissues)

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 EpiskinTM tissues were kept at room temperature in their packaging until required.
Description: The EpiskinTM 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.

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

Duration of post-treatment incubation (if applicable):

Triplicate tissues were treated with the test item for an exposure period of 15 minutes (± 1 minute).
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 replicates:

Triplicate tissues for test item, negative and positive controls

Type of coverage:

other: Not applicable

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

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 h 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:

3

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

4

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 1% 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	0.075	0.074	0.037	0.038	0.037	0.038	4
	2	0.068	0.068		0.031	0.031	0.031	3
	3	0.077	0.083		0.040	0.046	0.043	4

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	0.037	0.006	4	1

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

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

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 42h 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 42h of recovery period, the relative mean viability of the tissues treated with the test item was 4% with a standard deviation of 1% as assessed by the MTT assay. 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 Regulation (EC) No. 1272/2008 (CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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.
Transport from supplier to testing laboratory: The eyes were transported to testing laboratory 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) of test item was applied on each cornea
- Concentration (if solution): undiluted

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

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 pre-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 completion of the treatment period, the test and control items were removed from the front opening of the anterior chamber and the corneas were rinsed. The corneas were then incubated for 2 h ± 10 minutes in a water bath at 32 ± 1 °C and the second opacity measurement (OPT2) was performed.

Permeability determination
- Application of sodium fluorescein: After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution (4 mg/mL). The holders were then incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at 32 ± 1 °C for 90 ± 5 minutes. At the end of the incubation, the optical density at 490 nm (OD490) of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea.

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.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

93

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

20

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 20 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	-	-
	8	3	2	-1.0	-	0.021	-	-
	Mean	-	-	-1.0	-	0.009	-	-
	SD	-	-	0.0	-	0.010	-	-
Test item	45	2	0	-2.0	-1.0	0.106	0.097	0
	5	3	15	12.0	13.0	1.744	1.735	39
	38	3	16	13.0	14.0	0.439	0.430	20
	Mean	-	-	-	8.7	-	0.754	20
	SD	-	-	-	8.4	-	0.866	19.3
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

SD: standard deviation

OPT0: corneal opacity before treatment

OPT2: corneal opacity after the 2 h recovery period

Interpretation of results:

other: not predictable

Conclusions:

The ocular corrosive or severe irritant potential of test item dihydroterpineol 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 (no Category).
According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

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 dihydroterpineol 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). 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 20 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 (no Category).

According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July 2016

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: reconstructed human Cornea-like Epithelium

Details on test animals or tissues and environmental conditions:

EpiOcular TM tissue Model OCL-212 supplied by MatTek

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item DIHYDROTERPINEOL MULTICONSTITUENT was applied as supplied at the dose of 50 µL,

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

A 13-minute post exposure immersion period at room temperature and 2 hour and 7 minutes post exposure incubation at standard culture conditions.

Number of animals or in vitro replicates:

2 replicates.

Irritation parameter:

other: percent tissue viability

Value:

4.88

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

percent tissue viability = 22.84%

Irritant / corrosive response data:

DIHYDROTERPINEOL MULTICONSTITUENT has to be identified as potentially requiring classification and labelling according to UN GHS. Category 1 or 2 has to be defined based on the results of another eye irritation study.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

According to previous results of a study conducted according to OECD Guideline 437, the test item could not be identified as inducing serious eye damage (UN GHS Category 1). As a consequence, dihydroterpineol multiconstituent has to be classified in Category 2 according to UN GHS Regulation.

Executive summary:

In a GLP study conducted according to OECD Guideline 492, the irritant and corrosive potential of dihydroterpineol multiconstituent was evaluated. The method is based on viability of Reconstructed Human Cornea-like Epithelium-(RhCE) when exposed to the test substance. The mean percent tissue viability of the RhCE replicates treated with the test item was 4.88% versus 22.84 % in the positive control (Methyl acetate).

Therefore, dihydroterpineol multiconstituent has to be identified as potentially requiring classification and labelling according to UN GHS.

According to the results from a study conducted according to OECD Guideline 437, dihydroterpineol multiconstituent has to be classified in Category 2 according to UN GHS Regulation.

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^TMreconstructed 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 42h 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 treated tissues appeared white which was considered to be indicative of dead tissues. Following a 15 minutes exposure and 42h of recovery period, the relative mean viability of the tissues treated with the test item was 4% with a standard deviation of 1% as assessed by the MTT assay.

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 dihydroterpineol 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). 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 Vitro Irritancy Score (IVIS) for test item and positive control were 20 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 (no Category).

According to OECD Guideline 437, further testing should be conducted for classification and labeling purposes.

In a GLP study conducted according to OECD Guideline 492, the irritant and corrosive potential of Dihydroterpineol multiconstituent was evaluated. The method is based on viability of Reconstructed Human Cornea-like Epithelium-(RhCE) when exposed to the test substance. The mean percent tissue viability of the RhCE replicates treated with the test item was 4.88% versus 22.84 % in the positive control (Methyl acetate).

Justification for classification or non-classification

In an in vitro skin irritation study conducted according to OECD Guideline 439, the mean viability was < 50%. Therefore, the results met the criteria for an irritant response and the test material is classified in category 2 (H315) according to CLP Regulation (EC) No. 1272/2008.

According to previous results of a study conducted according to OECD Guideline 437, the test item could not be identified as inducing serious eye damage (UN GHS Category 1). As a consequence, dihydroterpineol multiconstituent is classified in Category 2 according to UN GHS Regulation.