Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Endpoint summary

Description of key information

Skin irritation/corrosion (OECD 439 and 431): not irritating

Eye irritation (OECD 437): 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 - 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:

adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU B.40 bis (In vitro Corrosion: Human Skin Model Test)

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™(EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23338
- Delivery date: 24 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 ± 0.5 min exposure); 37 ± 1.5 °C (60 ± 5 min exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were gently rinsed with DPBS 20 times in order to remove any residual test material.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, SoftMax Pro Enterprise v. 4.7.1)
- Wavelength: 570 nm
- Filter: without reference filter

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 2.078 ± 0.070 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 5.64 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance showed no reducing capacity 1 h after MTT incubation, an additional test with freeze-killed tissues was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than 50% and the viability after 1 hour exposure is greater than 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 50 µL

NEGATIVE CONTROL
- Amount applied: 50 µL

POSITIVE CONTROL
- Amount applied: 50 µL
- Concentration : 8 N

Duration of treatment / exposure:

3 ± 0.5 min and 60 ± 5 min

Number of replicates:

duplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

3 min exposure

Value:

97.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean value of 2 tissues

Run / experiment:

60 min exposure

Value:

105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance showed no reducing capacity 1 hour after MTT incubation.
- Colour interference with MTT: The test substance did not change colour, when mixed with deionised water and thus passed the colour interference pre-test.

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

Table 2. Results after 3 and 60 min treatment with the test substance and controls

	Exposure interval (min)	Absorbance of 3 wells		Aborbance at 570 nm *		Mean absorbance of 2 tissues	CV (%)	Rel. absorbance (% of negative control) **
		Tissue 1	Tissue 2	Tissue 1	Tissue 2
Negative control	3	1.738	1.636	1.701	1.598	1.649	4.4	100.0
Positive control		0.276	0.335	0.239	0.297	0.268	15.4	16.2
Test substance		1.624	1.672	1.587	1.635	1.611	2.1	97.7
Negative control	60	1.655	1.661	1.619	1.625	1.622	0.2	100.0
Positive control		0.234	0.185	0.198	0.148	0.173	20.2	10.7
Test substance		1.816	1.677	1.780	1.640	1.710	5.8	105.4

* Mean of three replicate wells after blank correction

 ** Relative absorbance (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Interpretation of results:

other: non-corrosive according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the reconstructed human epidermis test the test substance does not possess corrosive properties.

Executive summary:

The skin corrosion potential of the test substance was assessed by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2017). Each of two human skin tissues (EpiDerm™) with a tissue size of 0.63 cm² were treated with 50 µL of the test substance, the negative control (deionised water) or the positive control (potassium hydroxide 8 N) for 3 and 60 min. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 3 and 60 min treatment with the test substance compared to the negative control tissues was 97.7% and 105.4%, respectively. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 3 min and 15% after 60 min treatment the test substance was not considered to be corrosive to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, potassium hydroxide, revealed a mean cell viability of 10.7% (required ≤ 15%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was non-corrosive to the skin under the conditions of the test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb - 02 Jun 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:

adopted 28 Jul 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, 1st ATP 2009: EC Regulation No 761/2009 of 23 July 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm™ (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (EPI-200) (MatTek Corporation, Bratislava, Slovakia)
- Tissue batch number: 23395
- Delivery date: 21 Feb 2017
- Date of initiation of testing: 21 Feb 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C for 35 min in the incubator; thereafter at room temperature for 25 min in a sterile bench

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Tissues were gently rinsed with DPBS at least 15 times in order to remove any residual test material. After the rinsing the inserts were submerged in DPBS at least 3 times. Afterwards the inserts were once again rinsed with DPBS from the inside and the outside.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (Versamax, Molecular Devices, Softmax Pro v.4.7.1)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The quality of the EpiDerm tissue was assessed by undertaking an MTT cell viability test. The determined OD (540 - 570 nm) was 1.211 ± 0.175 (acceptance criteria: 1.0 - 3.0).
- Barrier function: The barrier function was assessed by determination of the exposure time required to reduce tissue viability by 50% (ET-50) upon application of 100 µL of 1% Triton X-100. The ET-50 value was determined to be 4.83 h (acceptance criteria: 4.77-8.72 h).
- Contamination: The cells used to produce the EpiDerm tissue were screened for the presence of viruses, bacteria, yeast and other fungi.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Since the test substance did not directly reduce MTT in a pre-test, an additional test with freeze-killed tissues was not performed.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after 1 hour exposure is less or equal to 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 30 µL

NEGATIVE CONTROL
- Amount applied: 30 µL

POSITIVE CONTROL
- Amount applied: 30 µL
- Concentration: 5% aqueous solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicates for each treatment and control group

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean of 3 tissues

Run / experiment:

60 min

Value:

88.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Direct-MTT reduction: The test substance did not interfere with the MTT assay (no reducing capacity).
- Colour interference with MTT: The test substance did not change colour when mixed with deionised water. Also its intrinsic colour was not intensive.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values (1.978, 1.710, 1.638) were in the range of the required acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 min treatment.
- Acceptance criteria met for positive control: Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1% thus confirming the validity of the test system.
- Acceptance criteria met for variability between replicate measurements: The relative standard deviations of the 3 identical replicates of the test substance, the positive and negative control, were < 18% (10.1, 4.1, 5.8%), thus ensuring the validity of the study.

Table 2. Results after treatment with the test substance and controls

Test group	Mean absorbance at 570 nm*			Rel. absorbance (%)**			SD (%)	Rel. absorbance (% of negative control)***
	Tissue 1	Tissue 2	Tissue 3	Tissue 1	Tissue 2	Tissue 3
Negative control	1.978	1.710	1.638	111.4	96.3	92.3	10.1	100.0
Positive control	0.057	0.056	0.053	3.2	3.1	3.0	4.1	3.1
Test substance	1.550	1.483	1.663	87.3	83.5	93.7	5.8	88.2

* Mean of three replicate wells after blank correction (blank = 0.036)

** Relative absorbance per tissue (rounded values): 100 × (absorbance tissue) / (mean absorbance negative control)

*** Relative absorbance per treatment group (rounded values): 100 × (mean absorbance test substance/positive control) / (mean absorbance negative control)

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions of the reconstructed human epidermis test the test substance does not possess any skin irritating potential.

Executive summary:

The skin irritation potential of the test substance was determined by an in vitro skin irritation test in human keratinocytes according to OECD Guideline 439 and in compliance with GLP (2017). 30 μL of the test substance, 30 µL of the negative control (DPBS) or 30 µL of the positive control (5% aqueous solution of sodium lauryl sulphate) were applied to triplicate tissues each and spread to match the surface of each tissue. The test substance and the positive and negative controls were washed off the skin tissues after 60 min treatment. After further incubation for about 42 h the tissues were treated with the MTT solution for 3 h following about 2.25 h extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1%, and thus assuring the validity of the test system. The test substance did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). After treatment with the test substance the mean relative absorbance value decreased to 88.2% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test substance is not considered to possess skin irritant potential.

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:

29 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

(July, 2013)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported to the laboratory in Hank's Buffered Salt Solution (HBSS) at ambient temperature.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
- Indication of any antibiotics used: not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.75 mL

POSITIVE CONTROL
- Amount applied: 0.75 mL

NEGATIVE CONTROL
- Amount applied: 0.75 mL

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

triplicates for each treatment and control group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS:
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. Each isolated cornea was mounted in a specially designed cornea holder.

TREATMENT METHOD:
The cornea holder 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 (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on the top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. After equilibration for about 1 hour, the anterior compartment received the test substance or the controls on the surface of the corneae. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath for 10 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS:
At the end of the equilibration period, the basal opacity was determined (t0). Each cornea with a value of a basal opacity > 7 was discarded.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE:
The test substance was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: 2 h in a vertical position

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea via an opacitometer (OP_KiT opacitometer, Electro Design, Riom France).
- Corneal permeability: The passage of sodium fluorescein dye was measured with the aid of a microplate reader (Versamax Molecular Devices) at 490 nm (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS), IVIS = opacity value + (15 x OD490 value)

DECISION CRITERIA:
Test substance with an IVIS > 55 was regarded as serious eye damage and labelled Category 1 according to CLP/EHS/GHS.
Test substance with an IVIS ≤ 3 was regarded as non-irritant and labelled in no category.
Test substance with an IVIS > 3; ≤ 55: no prediction can be made.

Irritation parameter:

in vitro irritation score

Remarks:

mean value of 3 corneae

Run / experiment:

10 min exposure

Value:

0.28

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.95).
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 82.07) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control resulted in opacity and permeability values that were less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.
- Acceptance criteria met for positive control: The positive control resulted in an IVIS which was within two standard deviations of the current historical mean.

Table 2. Results after 10 min incubation time.

Test group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS
		Mean		Mean
Negative Control	0	0.00	0.060	0.063	0.90	0.95
	0		0.066		0.99
	0		0.064		0.96
Positive Control	57.0*		1.074*		73.11	82.07
	66.0*		1.586*		89.79
	61.0*		1.498*		83.33
Test substance	0.00*		-0.004*		-0.07	0.28
	1.00*		-0.006*		0.91
	0.00*		0.001*		0.01

* Corrected values

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under conditions of the Bovine Corneal Opacity and Permeability Test (BCOP) the test subtance was not irritating to the eye. Application of the test substance to bovine corneae resulted in a calculated mean IVIS of 0.28.

Executive summary:

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (2016). After a first opacity measurement of the fresh bovine corneae, the neat test substance was applied directly to the epithelial surface of three cattle corneae in an incubation chamber in horizontal position for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae. Further, the corneae were incubated for another 120 min at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 0.28. With the negative control saline neither an increase of opacity nor permeability of the corneae could be observed. The mean IVIS of the negative control was 0.95. The mean IVIS of the positive control (2-ethoxyethanol) was 82.07. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

The skin corrosion potential of the test substance was assessed by an in vitro skin corrosion test using a human skin model according to OECD Guideline 431 and in compliance with GLP (2017). Each of two human skin tissues (EpiDerm™) with a tissue size of 0.63 cm² were treated with 50 µL of the test substance, the negative control (deionised water) or the positive control (potassium hydroxide 8 N) for 3 and 60 min. Cell viability was measured by dehydrogenase conversion of MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. The relative mean tissue viability obtained after 3 and 60 min treatment with the test substance compared to the negative control tissues was 97.7% and 105.4%, respectively. Since, the mean relative tissue viability for the test substance was above the threshold for irritancy of 50% after 3 min and 15% after 60 min treatment the test substance was not considered to be corrosive to the skin. The optical pre-experiment (colour interference pre-experiment) to investigate the colour change potential of the substance in water did not lead to a change in colour. Additionally, optical evaluation of the MTT-reducing capacity of the test substance after 1 hour incubation with MTT-reagent did not show blue colour. The positive control, potassium hydroxide, revealed a mean cell viability of 10.7% (required ≤ 15%) after 60 min exposure and thus ensuring the validity of the test system. All other acceptability criteria were met. Based on the results of this study, the test substance was non-corrosive to the skin under the conditions of the test.

The skin irritation potential of the test substance was determined by an in vitro skin irritation test in human keratinocytes according to OECD Guideline 439 and in compliance with GLP (2017). 30 μL of the test substance, 30 µL of the negative control (DPBS) or 30 µL of the positive control (5% aqueous solution of sodium lauryl sulphate) were applied to triplicate tissues each and spread to match the surface of each tissue. The test substance and the positive and negative controls were washed off the skin tissues after 60 min treatment. After further incubation for about 42 h the tissues were treated with the MTT solution for 3 h following about 2.25 h extraction of the colorant from the cells. The amount of extracted colorant was determined photometrically at 570 nm.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 3.1%, and thus assuring the validity of the test system. The test substance did not reduce MTT (pre-test for direct MTT reduction), and it did not change colour when mixed with deionised water (pre-test for colour interference). After treatment with the test substance the mean relative absorbance value decreased to 88.2% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test substance is not considered to possess skin irritant potential.

 

Eye

The eye irritation potential of the test substance was determined in a bovine corneal opacity and permeability test (BCOP test) according to OECD Guideline 437 and in compliance with GLP (2016). After a first opacity measurement of the fresh bovine corneae, the neat test substance was applied directly to the epithelial surface of three cattle corneae in an incubation chamber in horizontal position for 10 min at 32 ± 1 °C. After the incubation phase the test substance was rinsed from the corneae. Further, the corneae were incubated for another 120 min at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time. In addition, the permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 min at 32 ± 1 °C. The results of the opacity and permeability measurement of the test substance were used to calculate an in vitro irritation score (IVIS) of 0.28. With the negative control saline neither an increase of opacity nor permeability of the corneae could be observed. The mean IVIS of the negative control was 0.95. The mean IVIS of the positive control (2-ethoxyethanol) was 82.07. Based on the results, the test substance was not irritating to the eye under the conditions of the test.

Justification for classification or non-classification

The available data on skin and eye irritation of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.