rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Endpoint summary

Description of key information

Skin corrosion (OECD TG 431): not corrosive

Skin irritation (OECD TG 439): not irritating

Eye irritation (OECD TG 438): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 April 2016 - 27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Updated guideline adopted July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MatTek test protocol “In vitro EpiDerm(TM) Skin Corrosion Test (EPI-200-SCT)”

Version / remarks:

07 November 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 14 September 2015

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation (Bratislava, Slovakia)

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissue
- Tissue batch number(s): 23338
- Delivery date: 24 May 2016
- Date of initiation of testing: 24 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 3 minutes exposure: at room temperature, 60 minute exposure: 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times using a wash bottle containing DPBS
- Observable damage in the tissue due to washing: no

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The substance was checked for possible interference with the MTT endpoint before the start of the study. 50 μL of the test item was added to 0.3 mL of deionised water (transparent glass test-tube). The mixture was incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 % CO2) for 60 min. At the end of the exposure time, the mixture was shaken and the presence and intensity of the staining (if any) was evaluated. The test item did not dye water when mixed with it.
To test if an item directly reduces MTT, 50 μL of the test item were added to 1 ml of a MTT/DMEM solution (1 mg/mL) and were incubated in the incubator (37 ± 1.5 °C, 5 ± 0.5 CO2) for 60 minutes. Untreated MTT/DMEM solution (1 mg/mL) medium was used as control. The test item did not prove to be a MTT reducer.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Microplate reader: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 exposure times

EVALUATION
The mean OD of the duplicate negative control tissues was calculated after blank correction. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%)= [mean OD(test item/positive control)/mean ODnegative control] *100

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA:
1. The mean OD of the tissue replicates treated with the negative control should be ≥ 0.8 and ≤ 2.8 for every exposure time
2. The mean viability of the tissue replicates treated with the positive control for 1 hour, should be <15% compared to the negative control
3. The Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates should be ≤ 30%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μL (79.4 μL/cm^2)

Duration of treatment / exposure:

3 +/- 0.5 minutes and 60 +/- 0.5 minutes

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure / mean of 2 replicates

Value:

97.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

16.2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure / mean of 2 replicates

Value:

105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

10.7

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean OD of the tissue replicates treated with the negative control is ≥ 0.8 and ≤ 2.8 for every exposure time (values between 1.627 and 1.755)
- Acceptance criteria met for positive control: yes, the mean viability of the tissue replicates treated with the positive control for 1 hour, is <15% (10.7%) compared to the negative control
- Acceptance criteria met for variability between replicate measurements: yes, the Coefficient of Variation (CV) in the range 20 – 100% viability between tissue replicates is ≤ 30% (values between 0.2% and 5.8%)

Mean OD₅₇₀ values and viabilities for the negative control, positive control and test item are given below:

Item	Exposure Period (minutes)	Individual OD570 of Individual tissues	Mean OD570 of duplicate tissues (tvt)	Water-Killed Tissues			True Viability tvt-(tkt-ukt)	Relative mean viability (%)
				tkt	ukt	tkt-ukt
Negative Control Item	60	1.634	0.818					100*
		1.639
Positive Control Item		0.219	0.027					10.7
		0.186
Test Item	3	1.587	1.611					97.7
		1.635
	60	1.780	1.710					105.4
		1.640

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

Interpretation of results:

other: Not corrosive to the skin

Remarks:

In accordance with EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The results of an in vitro skin corrosion test showed that the substance was not corrosive to the skin (tissue viability after 3 minutes exposure: 97.7% and tissue viability after 60 minutes exposure: 105.4%).

Executive summary:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (deionised water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met. The cell viability of the tissues exposed to the substance were 97.4% and 105.4% for 3 minutes and 60 minutes exposure, respectively. Both values did not exceed thereshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), therefore the substance is considered not to be corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 February 2017 - 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 14 September 2015

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation (82105 Bratislava, Slovakia)

Source strain:

other: not applicable

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Epi-200- SIT Kit (MatTek)
- Tissue lot number: 23395
- Delivery date: 21 February 2017
- Date of initiation of testing: 21 February 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: standard culture incubation conditions
- Temperature of post-treatment incubation: standard culture incubation conditions

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed with DPBS (>= 15 washing steps), submerged in DPBS at least three times and washed once more with sterile DPBS from the inside and outside. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with sterile blotting paper.
- Observable damage in the tissue due to washing: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE:
- Incubation time: 3 hours
- Measurement method: microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter
- MTT concentration: 1 mg/mL
- The test item was checked for interference with the MTT endpoint before the start of the study.

NUMBER OF REPLICATE TISSUES: 3 for the test substance, the negative and the positive control each.

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

CELL VIABILITY MEASUREMENT: After incubation, tissues were incubated with 0.3 mL MTT-solution for 3 hours. After incubation, tissues were rinsed and transferred into a 24-well plates containing 2 mL of isopropanol. Formazan salt was extracted for 2.25 hours while shaking at room temperature. After the extraction period, inserts were pierced with an injection needle to allow the extract to run off. Inserts were discarded and the 24-well plates were placed on a shaker for 15 minutes until the solution was homogeneous in colour. Per tissue, 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate from the 15 minutes exposure. OD was read in a microplate reader (Versamax® Molecular Devices, Softmax Pro, version 4.7.1) with a 570 nm filter. Mean values were calculated from the 3 wells per tissue.

PREDICTION MODEL / DECISION CRITERIA: see Table 1

ACCEPTABILITY CRITERIA
a) The absolute OD 570 nm of the negative control tissues should be ≥ 0.8 and ≤ 2.8.
b) The mean relative tissue viability of the positive control should be ≤20%.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST ITEM:
- Amount applied: 30 μL (47 μL/cm^2), spread to match the surface of the tissue

CONTROLS:
- Amount applied: 30 μL

Duration of treatment / exposure:

60 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates (3 tissues treated with the test item, the positive control and the negative control, each)

Irritation / corrosion parameter:

% tissue viability

Remarks:

Mean of three tissues

Run / experiment:

60 minutes exposure

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability: 3.1%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval (values between 1.674 and 2.014).
- Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1%.
- The relative standard deviations between the % variability values of the test item, the positive and negative controls in the main test were below 11%.

- Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.

Table 2 Summary of results

Dose group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean absorbance of 3 wells	Mean absorbance of three wells blank corrected	Mean absorbance of 3 tissues after blank correction	Relative absorbance (%) tissue 1, 2 + 3	Relative Standard Deviation (%)	Mean relative absorbance (% of negative control)
Blank		0.036	0.036	0.036	0.036	0.000
Negative control	1	2.032	1.993	2.017	2.014	1.978	1.775	111.4	10.1	100.0
	2	1.749	1.741	1.746	1.746	1.710		96.3
	3	1.681	1.676	1.666	1.674	1.638		92.3
Positive control	1	0.085	0.106	0.089	0.093	0.057	0.055	3.2	4.1	3.1
	2	0.093	0.091	0.092	0.092	0.056		3.1
	3	0.089	0.088	0.089	0.089	0.053		3.0
Test item	1	1.590	1.590	1.576	1.586	1.550	1.565	87.3	5.8	88.2
	2	1.510	1.534	1.513	1.519	1.483		83.5
	3	1.701	1.696	1.701	1.699	1.663		93.7

Interpretation of results:

other: Not a skin irritant

Remarks:

According to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The substance is not irritant to the skin (88.2% tissue viability after 60 minutes of treatment).

Executive summary:

An in vitro study was performed according to OECD 439 and GLP principles, to assess the substance's potential to be irritant to the skin. Three tissues from the human skin model EpiDerm(TM) were exposed to the substance, a positive control and a negative control for 60 minutes. Before the start of the study, the test item was checked for interference with the MTT endpoint. Tissues were washed and incubated with MTT after the exposure period. Formazan was extracted by isopropanol and subsequently the tissues were read by a microplate reader at 570 nm. The positive control tissues showed a mean tissue viability of 3.1% indicating that the test system functioned properly. The negative control tissues had absorbance values within the required acceptability criteria. Since all acceptability criteria were met, the study was considered to be valid. The tissues treated with the substance showed a mean tissue viability of 88.2% compared to the negative control tissues. Based on this result, the substance is not irritant to the skin.

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:

28 Mar 2017 to 15 Mai 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

26 July 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist

Species:

other: Eyes of male or female chickens (ROSS, spring chickens)

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 μL neat substance

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Test group and positive control: triplicates
Negative control: Singlo

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline

Irritation parameter:

percent corneal swelling

Run / experiment:

slit-lamp examination

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

cornea opacity score

Run / experiment:

slit-lamp examination

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Irritation parameter:

fluorescein retention score

Run / experiment:

slit-lamp examination

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: maximum mean values

Other effects / acceptance of results:

Slit-lamp examination: The test substance caused corneal effects consisting of very slight corneal swelling (mean of 3%), no opacity and very slight fluorescein retention (mean score of 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities as well. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis.

Interpretation of results:

other: Not an eye irritant.

Remarks:

According to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant.

Executive summary:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 3%), no opacity and very slight fluorescein retention (mean score of 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities as well. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis. Based on these results, the substance is not considered to be irritating to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin corrosion:

The substance was tested in duplicate in an in vitro skin corrosion test according to OECD TG 431 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (deionised water) and a positive control (8.0 N KOH) for 3 minutes and 60 minutes. The substance was tested for direct MTT reduction and colour interference and both results were negative. Acceptability criteria for the negative control, positive control and variability between measurements were met.

The cell viability of the tissues exposed to the substance was 97.4% and 105.4% for 3 minutes and 60 minutes exposure, respectively. Since the cell viabilities did not exceed the threshold for corrosivity (50% after 3 minutes exposure and 15% after 60 minutes exposure), the substance is considered not to be corrosive.

Skin irritation:

An in vitro study was performed according to OECD 439 and GLP principles, to assess the substance's potential to be irritant to the skin. Three tissues from the human skin model EpiDerm(TM) were exposed to the substance, a positive control and a negative control for 60 minutes. Before the start of the study, the test item was checked for interference with the MTT endpoint.

Tissues were washed and incubated with MTT after the exposure period. Formazan was extracted by isopropanol and subsequently the tissues were read by a microplate reader at 570 nm. The positive control tissues showed a mean tissue viability of 3.1% indicating that the test system functioned properly. The negative control tissues had absorbance values within the required acceptability criteria. Since all acceptability criteria were met, the study was considered to be valid. The tissues treated with the substance showed a mean tissue viability of 88.2% compared to the negative control tissues. Based on this result, the substance is not irritant to the skin.

Eye irritation:

The test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) test, in accordance with OECD guideline 438 and GLP. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL 5% Benzalkonium Chloride (BAC)) were tested. After the exposure, the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. Mean fluorescein retention score was determined at 30 minutes post-treatment. The test substance caused corneal effects consisting of very slight corneal swelling (mean of 3%), no opacity and very slight fluorescein retention (mean score of 0.5). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance did not reveal any abnormalities. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities as well. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight or severe erosion and very slight or slight vacuolation of the epithelium and endothelial necrosis. Based on these results, the substance is not considered to be irritating to the eyes.

Justification for classification or non-classification

Based on the results, the substance does not need to be classified for corrosion, skin irritation or eye irritation according to EU CLP (EC No. 1272/2008 and its amendments).