Reaction Mass of Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.R,1R,6S)- and Cyclohexanepropanol, 2,2,6-trimethyl-a-propyl-, (alpha.S,1R,6S)-

Endpoint summary

Description of key information

Skin irritation/corrosion: not irritating (OECD 439, GLP, K, rel. 1).

Eye irritation: not irritating (OECD 437, GLP, K, rel. 1).

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:

From February 18 to 24, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 439 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

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:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit, SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 14-EKIN-005
- Production date: 18 February 2014
- Shipping date: 18 February 2014
- Delivery date: 18 February 2014
- Date of initiation of testing: 18-24 February 2014
- Expiry date: 24 February 2014

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Not reported. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/L
- Incubation time: 3 hours
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nm
- Filter: without reference filter
- Filter bandwidth: not applicable
- Linear OD range of spectrophotometer: not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function: 2.2 mg/mL ( ≥ 1.5 mg/mL)
- Morphology: well differenciated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum
- Contamination:
Absence of HIV1 and 2 antobodies, hepatitis C antibodies, hepatitis B antigen HBs
Absence of bacteria, fungus and mycoplasma
- Reproducibility: not reported

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE : the test item did not directly reduce MTT

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is less or equal to 50%
- The test substance is considered to be non-irritant to skin if the viability after the 15-min exposure period followed by the 42h post-exposure period is greater than 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): undiluted

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): undiluted

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% w/v

Duration of treatment / exposure:

15 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

63.7

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: none
- Direct-MTT reduction: the test item was not able to directly reduce MTT
- Colour interference with MTT: no colour interference were observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied.
- Acceptance criteria met for variability between replicate measurements:
- Range of historical values if different from the ones specified in the test guideline: The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.
For the previous 35 experiments conducted between May 2013 and February 2014 using this test method, the mean OD of the positive control was 0.084 ± 0.036 and the mean percentage viability was 9.6 ± 4.4. In this same period the mean OD of the negative control was 0.898 ±0.106.
The positive control reflected the ability to respond to an irritant test item under the conditions of the test. The negative control in this study was greater than the mean OD of the negative control in the previous 35 experiments conducted with this test method.

Table 7.3.1/1: Mean OD562 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

 

Item	OD562 of tissues	Mean OD562 of triplicate tissues	± SD of OD562	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.034	1.013	0.024	102.1	100*	2.4
	1.018			100.5
	0.986			97.3
Positive Control Item	0.062	0.054	0.011	6.1	5.3	1.1
	0.042			4.1
	0.058			5.7
Test Item	0.687	0.646	0.085	67.8	63.7	8.4
	0.702			69.3
	0.548			54.1

SD=        Standard deviation

*=         The mean viability of the negative control tissues is set at 100%

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 63.7% after the 15‑minute exposure period and 42 hours post‑exposure incubation period.

 

The relative mean tissue viability for the positive control treated tissues was 5.3% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1%. The positive control acceptance criterion was therefore satisfied. The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the percentage viability was 2.4%. The negative control acceptance criterion was therefore satisfied. The standard deviation calculated from individual percentage tissue viabilities of the three identically treated test item tissues was 8.4%. The test item acceptance criterion was therefore satisfied.

 

Under the test conditions, test item was classified as non-irritant according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

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:

March 11, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 437 without any deviation.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2013

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Number of animals: not mentioned
- Characteristics of donor animals (e.g. age, sex, weight): adult (12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported to the test facility over ice packs on the same day of slaughter
- Time interval prior to initiating testing: The corneas were prepared immediately on arrival
- indication of any existing defects or lesions in ocular tissue samples: None
- Indication of any antibiotics used: None

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): 0.75 mL was applied on each cornea
- Concentration: Undiluted

Duration of treatment / exposure:

10 minutes at 32 ± 1 °C

Observation period (in vivo):

- Corneal opacity was measured pre-treatment, post-treatment and post-incubation (after 120 minutes of incubation).
- Application of Sodium Fluorescein (4 mg/mL) and corneal permeability was measured after 90 min of incubation at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

120 minutes at 32 ± 1 °C.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitomete. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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

NEGATIVE CONTROL USED
0.9% w/v sodium chloride solution. Batch 300999 104. Purity: 0.9%. Expiry: 01 January 2015.

POSITIVE CONTROL USED
Ethanol. Batch SZBA0290. Expiry: 06 August 2014.

APPLICATION DOSE AND EXPOSURE TIME
- Application:0.75 mL of the test item or control items
- Exposure time: 10 minutes at 32 ± 1 ºC

TREATMENT METHOD: not reported

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

POST-EXPOSURE INCUBATION: 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas.
The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of Anthos 2001 microplate reader (OD492)
- Others: The corneas were retained after testing for possible conduct of histopathology

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean 3 corneas

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS = 2.3

Positive controls validity:

valid

Remarks:

IVIS = 46.7

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: not included

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied
- Acceptance criteria met for positive control: The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0. The positive control acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: not reported

Table 7.3.2/1: Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number		Opacity				Permeability (OD)		In VitroIrritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	7	2	3	5	3		0.052
	18	2	2	2	0		0.035
	9	2	2	4	2		0.047
					1.7*		0.045¨		2.3
Positive Control	4	2	24	25	23	21.3	1.822	1.777
	5	3	23	24	21	19.3	1.516	1.471
	6	1	24	29	28	26.3	1.664	1.619
						22.3·		1.623·	46.7
Test Item	1	2	3	3	1	0.0	0.018	0.000
	2	1	1	1	0	0.0	0.019	0.000
	3	1	1	1	0	0.0	0.038	0.000
						0.0·		0.000·	0.0

OD= Optical density         * = Mean of the post-incubation -pre‑treatment values        ¨= Mean permeability                           ·= Mean corrected value

Table 7.3.2/2: Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	7	clear	clear
	18	clear	clear
	9	clear	clear
Positive Control	4	cloudy	cloudy
	5	cloudy	cloudy
	6	cloudy	cloudy
Test Item	1	clear	clear
	2	clear	clear
	3	clear	clear

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions, with an IVIS < 3, test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 120 minutes.Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). 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). 

The test item, negative and positive control induced an IVIS of 0.0, 2.3 and 46.7, respectively.

The corneas treated with the negative control and test item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0, therefore the acceptance criterion was satisfied. The negative control gave opacity of ≤4.7 and permeability ≤0.080, therefore the acceptance criterion was satisfied.

Under the test conditions, with an IVIS < 3, test item does not require classification for eye irritation or serious eye damage according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

A key study was identified (Harlan, 2014).The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

This test was designed to be compatible with the Method B.46 of Commission Regulation (EC) No. 440/2008/EC and was performed in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the test item treated tissues was 63.7 ± 8.4 %, after the 15‑minute exposure period.

With a tissue viability > 50%, the test material was not considered to be irritant to skin.

Eye irritation:

A key study was identified (Harlan, 2014). This study was performed to assess the ocular irritancy potential of the undiluted test item to the isolated bovine cornea. The study was conducted according to the OECD guideline No. 437 and in compliance with GLP. The quality criteria required for acceptance of results in the test were satisfied. The In Vitro Irritancy Score of the test item was 0, after the 10 -minute exposure period followed by 120 -minute incubation period.

With an IVIS < 3, the test item does not require classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

 

Self-classification:

Based on the available information no self-classification is proposed regarding both skin and eye irritation according to the CLP and to the GHS.

No data was available regarding respiratory irritation, however the substance not being classified for skin and eye irritation, no classification is expected for respiratory irritation.