Cyclohexanol, 4-(1,1-dimethylethyl)-, trans-

Endpoint summary

Description of key information

Skin irritation: not irritating (EU method B.4; GLP compliant)
Eye irritation: not corrosive/severely eye irritating (EU method B.47; GLP compliant).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03-18 to 2008-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Minor deviations from the guidelines OECD 404 (2002) and EU Method B.4 (2008) - the stability of the test material were missing in the study report. - according to the guidelines, the body weight of the animals at the end of the study should be stated. This information was missing in the study report.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

2008

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002-04-24

Deviations:

yes

Remarks:

please refer to "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2007-10-15

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan France SARL, 03800 GANNAT, France
- Age at study initiation: twelve to twenty weeks old
- Weight at study initiation: 2.0 to 3.5 kg
- Housing: the animals were individually housed in suspended cages.
- Diet (ad libitum): Certified Rabbit Diet
- Water (ad libitum): mains drinking water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 17 to 23°C
- Relative humidity: 30 to 70%
- Air exchanges: at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

semiocclusive

Preparation of test site:

other: clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): a quantity of 0.5 g of the test material, moistened with the vehicle

VEHICLE
- Amount(s) applied (volume or weight with unit): 0.5 mL of distilled water

Duration of treatment / exposure:

4 hours

Observation period:

approximately one hour following the removal of the patches, and 24, 48 and 72 hours later

Number of animals:

3 male rabbits

Details on study design:

TEST SITE
- Area of exposure and type of wrap used: on the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.
On the day of the test a suitable test site was selected on the back of each rabbit.
A quantity of the test material moistened with the vehicle, was introduced under a 2.5 cm X 2.5 cm cotton gauze patch and placed in position on the shorn skin. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing: the corset and patches were removed from each animal and any residual test material removed by gentle swabbing with cotton wool soaked in distilled water.
- Time after start of exposure: four hours after application

SCORING SYSTEM: according to the Draize scale
Any other skin reactions, if present, were also recorded.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

No evidence of skin irritation was noted during the study.
No corrosive effects were noted.

Interpretation of results:

not irritating

Conclusions:

The test item is not irritating to the skin.
According to 67/548/EC and subsequent amendments, the test item is not classified as a skin irritant.
According to the EC Regulation No. 1272/2008 and subsequent amendments, the test item is not classified as a skin irritant.

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:

2011-08-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study reliable without restrictions Deviations from the guideline without an impact on the outcome of this study: According to the OECD guideline, for surfactants (means with a surface tension < 60 mN/m) a different treatment protocol should be used (e.g. tested at concentration of 10% w/v test item solution instead of 20% w/v and reduced exposure time for treatment of cornea are needed). Although the tested substance has surface active properties (41.9 mN/m), the protocol for a non surface active solid substance was used for test performance. However, it is explicitly stated that this has no impact on the outcome of this study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 2009

Deviations:

yes

Remarks:

please refer to the "Rationale for reliability incl. deficiencies" above

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

9.12.2010

Deviations:

yes

Remarks:

please refer to the "Rationale for reliability incl. deficiencies" above

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Details on test animals or tissues and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals.

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL of the test item was applied
- Concentration (if solution): 20% (w/v) suspension in saline (0.9% (w/v) NaCl in deionised water) of the test item

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

not applicable

Number of animals or in vitro replicates:

not applicable

Details on study design:

PREPARATION OF THE INCUBATION MEDIA
The medium MEM (Minimum Essential Medium), supplemented with sodium bicarbonate and L-glutamine was prepared one day prior to the start of the assay, and stored in a refrigerator (2 - 8 °C).
Immediately before starting the test, MEM was supplemented with 1% foetal calf serum (FCS). Afterwards, it was called cMEM (complete medium).
The OECD foresees the use of EMEM (Eagle’s minimal essential medium) which is in composition and osmolarity equivalent to the cMEM, thus cMEM could be used without restriction.

COLLECTION OF BOVINE EYES
Freshly isolated bovine eyes from at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s BSS supplemented with streptomycin / penicillin at ambient temperature. The corneas were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine, Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

PREPARATION OF CORNEAE
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above.
Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OEDC guideline 437, annex III, that consists of anterior and posterior compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (Oring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the complete medium was removed from both compartments and replaced by fresh cMEM, and the basal opacity was determined (t0).

OUTLINE OF STUDY
The anterior compartment received the test item or negative control (saline (produced in-house, lot no. 25.6.11)) or positive control (10% (w/v) Benzalkonium chloride (Sigma, Steinheim, Germany, lot no. 036K0208) in 0.9% (w/v) NaCl in deionised water) at a volume of 0.75 mL each on the surface of the corneas and was incubated at 32 ± 1 °C in the waterbath in a horizontal position. The test item, positive control and negative control were tested in triplicate.
Prior to the application the test item was melted up to 75 °C in the water bath and suspended in saline (20% (w/v)). The positive control was 10% (w/v) Benzalkonium chloride in saline. Saline was used as negative control item.
According to the OECD guideline, for surfactants (means with a surface tension < 60 mN/m) a different treatment protocol should be used (e.g. tested at concentration of 10% w/v test item solution instead of 20% w/v and reduced exposure time for treatment of cornea are needed).
Although the tested substance SymSitive® 1609 pur has surface active properties (< 60 mN/m), the protocol for a non surface active solid substance was used for test performance. However, it is explicitly stated that this has no impact on the outcome of this study.
The incubation time lasted 240 minutes (± 5 minutes).
After the test item or control items, respectively, were rinsed off for three times from the application side with saline. No visual evidence of the test item could be observed afterwards. Fresh cMEM was added into the anterior compartment and opacity was measured (t240). There was no need to add phenol red to the medium as a second marker for successful rinsing since the washing steps were performed very stringently.
In the second step of the assay, permeability of the cornea was determined. 1 mL of a Nafluorescein solution, 0.5 % (w/v) dissolved in HBSS (Hank’s buffered salt solution), was placed in the anterior compartment. Corneas were incubated again in a horizontal position for an additional 90 minutes at 32 ± 1 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

OPACITY MEASUREMENT
The basal opacity of all corneas was recorded using an opacitometer. Each corneas with a value of the basal opacity > 7 was discarded.
After the incubation time of 240 minutes with the test item, positive control and negative control the opacity was measured again (t240).

PERMEABILITY DETERMINATION
The permeability endpoint was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and the optical density at 490 nm (OD490) was determined with a spectrophotometer.

CRITERIA FOR DETERMINATION OF A VALID TEST
The test was acceptable if the in vitro irritation score of the positive control was ≥ 30 and the in vitro irritation score of the negative control was ≤ 3.

EVALUATION OF RESUTLS
- Opacity: the change of opacity value of each treated cornea or positive and negative control corneas was calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae was calculated and this value was subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
- Permeability: the corrected OD490 value of each cornea treated with positive control and test item was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.

IN VITRO SCORE CALCULATION
The following formula was used to determine the in vitro irritation score of the negative control:
In vitro Irritation Score = opacity value + (15 x OD490 value)
The following formula was used to determine the in vitro irritation score of the positive control and the test item:
In vitro Irritation Score = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The in vitro irritation score was calculated for each individual treatment and positive control cornea. The mean in vitro irritation score irritation value of each treated group was calculated from the individual in vitro irritation score values. Depending on the score obtained, the test item was classified into the category according to OECD guideline 437 (table 1 in the field "Any other information on materials and methods incl. tables" below).

Irritation parameter:

cornea opacity score

Run / experiment:

negative control

Value:

1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

positive control

Value:

266

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

test item

Value:

0

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

test item

Value:

0.33

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

positive control

Value:

287.24

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

negative control

Value:

2.12

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Table 1: Results after 240 minutes incubation time

Test group	Opacity value = Difference (t240 – t0) of Opacity		Permeability at 490 nm (OD490)		In vitro Score	Mean in vitro irritation score	Proposed in vitro Irritation Scale
		Mean		Mean
Negative control	1	1.00	0.077	0.075	2.16	2.12	Non corrosive /non severe irritant
	1		0.073		2.10
	1		0.074		2.11
Positive control	191.00*		0.939*		205.09	287.24	Corrosive /severe irritant
	377.00*		2.390*		412.86
	230.00*		0.917*		243.76
SymSitive®1609 pur	1.00*		0.039*		1.59	0.33	Non corrosive /non severe irritant
	-1.00*		0.029*		-0.56
	0.00*		-0.003*		-0.04

* corrected values

 

- with the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 2.12).

-the positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean in vitro irritation score 287.24) corresponding to a classification as corrosive /severe irritant to the eye (CLP/EPA/GHS (Cat 1)).

 

 

Table 2: Historical Data

	Positive control	Negative control
Mean in vitro Irritation Score	162.65	1.70
Standard Deviation	38.05	0.79

 

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not corrosive / not severely irritating to the eye (CLP (Cat 1)).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

One fully reliable animal study described in Bradshaw (2008) (EU method B.4; GLP compliant) was considered. The substance was not determined to be a skin irritant.

Eye irritation

One reliable in vitro study described by Heppenheimer (2012) (EU method B.47; GLP compliant) is considered to be reliable without restrictions. The substance was determined not to be corrosive or severly irritating to the eyes.

Justification for selection of skin irritation / corrosion endpoint:
One fully reliable animal study described in Bradshaw (2008) (EU method B.4; GLP compliant) was considered. The substance was not determined to be a skin irritant.

Justification for selection of eye irritation endpoint:
One reliable in vitro study described by Heppenheimer (2012) (EU method B.47; GLP compliant) is considered to be reliable without restrictions. The substance was determined to be not corrosive or severly irritating to the eyes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008

 

Skin irritation

An in vivo skin irritation study (2008) is considered as the key study and will be used for classification. The skin irritation was scored according to the Draize scale. The mean score (24, 48, 72h) for erythema and oedema was 0 for all animals. The study was terminated after the 72 hour observation. According to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) No 2021/849, the substance will not be classified as skin irritant.

 

Eye irritation

An in vitro eye irritation study (2012) is considered as the key study and will be used for classification. According to this study, the mean in vitro score of the in vitro bovine corneal opacity and permeability assay (BCOP, according to EU method B.47) was 0.33. The value was below the threshold value of 55.1 for severe eye irritants . The classification criteria according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) No 2021/849 as severe eye irritant are not met, hence no classification is required.

 

Respiratory irritation

The classification as respiratory irritant is normally covered under the endpoint specific target organ toxicity- single exposure and repeated exposure. Please refer to the endpoint summaries on acute toxicity (endpoint 7.2) for further information.