1,1-diethoxyethane

Endpoint summary

Description of key information

Skin irritation/corrosion: Under the conditions of the in vitro study, the test item is considered to possess a corrosive potential to skin (UN GHS Category 1) (reference 7.3.1-1).

The available in vivo reports indicate a skin irritating potential of the test item (reference 7.3.1-2 to 7.3.1-5).

Eye irritation: In a study according to OECD TG 437 the test item is considered to cause serious eye damage (reference 7.3.2-1). In a study performed according to OECD 492 no prediction on the eye irritating potential of the test item could be made (reference 7.3.2-2).

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:

weight of evidence

Study period:

from 2018-09-04 to 2019-01-10

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: B.40.bis. In vitro skin corrosion: human skin model test

Version / remarks:

Council Regulation (EC) No. 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic™ Skin Corrosivity Test (2012)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstituted human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i. e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 18-RHE-142
- Date of initiation of testing: On day of receipt the pre-incubation phase of the tissues started.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 20 mL DPBS; excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 10

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin category 1 if the viability is more than or equal to 50 % after 3 min and less than 15 % after 1 h exposure or less than 50 % after 3 min exposure.
- The test substance is considered to be non-corrosive to skin if the viability is greater than or equal to 50 % after 3 min and greater than 15 % after 1 h exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Solid test item: 40 ± 3 µL per tissue
Positive control: 40 ± 3 µL per tissue
Negative control: 40 ± 3 µL per tissue

Duration of treatment / exposure:

test item and negative control - 3 min and additional 1 hour
positive control - only 1 hour

Number of replicates:

test item: 2 tissues per time point (3 min and 1 hour)
negative control: 2 tissues per time point (3 min and 1 hour)
positive control: 2 tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item after 3 min exposure

Value:

>= 50

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item after 1 h exposure

Value:

< 15

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

THER EFFECTS
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Following treatment with the test item, the tissue viability was >50 % after 3 minutes exposure (mean viability: 100.8 %) and <15 % after 1 hour exposure (mean viability: 9.8 %), i.e. according to OECD 431 the test item is considered as corrosive to skin (UN GHS Category 1).

Table 1. Results after treatment of the RHE-model with the test item

Group		Tissue 1		Tissue 2		Mean		CV
		OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	3 min	1.813	97.9 %	1.891	102.1 %	1.852	100.0 %	3.0 %
	1 hour	1.640	103.9 %	1.515	96.0 %	1.578	100.0 %	5.6 %
Positive Control	1 hour	0.013	0.8 %	0.009	0.6 %	0.011	0.7 %	14.3 %
Test item	3 min	1.806	97.5 %	1.926	104.0 %	1.866	100.8 %	4.6 %
	1 hour	0.166	10.5 %	0.144	9.1 %	0.155	9.8 %	10.2 %

 

Interpretation of results:

Category 1 (corrosive) based on GHS criteria

Conclusions:

Under the conditions of the study, the test item is considered to possess a corrosive potential to skin (UN GHS Category 1).

Executive summary:

An in vitro study in accordance with the OECD Guideline OECD 431 and EU Method B.40.bis was performed to assess the skin corrosion potential of the test item. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and 1 hour. The positive control was treated only for 1 hour. 40 ± 3 µL of either the negative control (deionised water), the positive control (potassium hydroxide, 8N) or the test item were applied to the tissues.

The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.7 % and, thus, lower than the historically established threshold of 1.01 %. After treatment with the negative control (deionised water) the mean ODs were 1.852 (3 minutes exposure) and 1.578 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.587 and 1.422, respectively.

Following treatment with the test item, the tissue viability was >50 % after 3 minutes exposure (mean viability: 100.8 %) and <15 % after 1 hour exposure (mean viability: 9.8 %), therefore, the test item is considered as corrosive to skin (UN GHS Category 1).

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

other: Data collection

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline required

Principles of method if other than guideline:

- Principle of test: Data collection

GLP compliance:

not specified

Species:

rabbit

Amount / concentration applied:

500 mg

Duration of treatment / exposure:

24 h

Irritation parameter:

other: Descriptive

Basis:

mean

Time point:

24 h

Remarks on result:

probability of mild irritation

Interpretation of results:

study cannot be used for classification

Executive summary:

In the review article, mild irritation was reported after application of 500 mg test item onto rabbit skin for 24 h.

Reference 3

Endpoint:

skin irritation: in vivo

Type of information:

other: Data collection

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline followed

Principles of method if other than guideline:

Application of the test item onto the prepared skin of rabbit.

GLP compliance:

not specified

Species:

rabbit

Amount / concentration applied:

10 mg

Duration of treatment / exposure:

24 h

Irritation parameter:

other: Descriptive

Basis:

mean

Time point:

24 h

Remarks on result:

probability of weak irritation

Interpretation of results:

study cannot be used for classification

Executive summary:

In the review article, weak irritation was reported after application of 10 mg test item onto rabbit skin for 24 h.

Reference 4

Endpoint:

skin irritation: in vivo

Type of information:

other: Data collection

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline required

Principles of method if other than guideline:

- Principle of test: data collection

GLP compliance:

not specified

Species:

rabbit

Number of animals:

5

Irritation parameter:

other: similar to Draize et al., 1944

Basis:

mean

Score:

2

Max. score:

10

Reversibility:

not specified

Remarks on result:

probability of weak irritation

Interpretation of results:

study cannot be used for classification

Executive summary:

In the review article, weak irritation was reported after application of the test item onto rabbit skin.

Reference 5

Endpoint:

skin irritation: in vivo

Type of information:

other: Review article

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Qualifier:

no guideline required

Principles of method if other than guideline:

- Principle of test: Review article

GLP compliance:

not specified

Species:

rabbit

Type of coverage:

occlusive

Preparation of test site:

other: intact or abraded skin

Duration of treatment / exposure:

24 h

Irritation parameter:

other: Descriptive

Basis:

mean

Time point:

24 h

Remarks on result:

no indication of irritation

Interpretation of results:

study cannot be used for classification

Executive summary:

In the review article, no irritation was reported after application of the test item onto rabbit skin under occlusion for 24 h.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-08-10 to 2018-08-17

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse, Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age of the cattle: 15 - 53 months
- Corneal diameter: 25 - 28 mm

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: The liquid test item (Acetal) was tested undiluted

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Incubation for 120 minutes after treatment. For permeability determination corneas were incubated for additional 90 minutes.

Number of animals or in vitro replicates:

Three corneas were used per group (negative control, positive control or test item group).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated corneas were used. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, 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 incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : 0.9 % sodium chloride solution

POSITIVE CONTROL USED : N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of undiluted liquid test item for 10 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed at least three times with wash medium, final rinse with incubation medium
- POST-EXPOSURE INCUBATION: Incubation for 120 minutes after treatment. For permeability determination corneas were incubated for additional 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values. From the individual corrected opacity values, a mean corrected opacity value was calculated for each group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were
<3 No Category
>3; <55 No prediction can be made
>55 Category 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

62.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.3 - 5.5). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 100.0 (study acceptance criteria range: 77.1 - 119.4). Therefore, the study fulfilled the acceptance criteria.

Table 1: The IVIS obtained after treatment

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9 % sodium chloride solution	2.1	0	2.1	1.1	0.9
		0.3	-0.001	0.285
		0.9	0.002	0.93
Positive control	N,N-dimethyl-formamide	90.5	0.715	101.225	100	4.5
		74.7	1.94	103.8
		79.3	1.051	95.065
Test item	Art. W200220	33.4	1.768	59.92	62.8	3.1
		31.2	2.322	66.03
		27.2	2.359	62.585

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In a study accoring to OECD TG and EU Method the test item was determined to be eye irritating (Category 1 (irreversible effects on the eye) based on GHS criteria).

Executive summary:

To assess the ocular corrosiveness and eye irritating properties a GLP-compliant Bovine Corneal Opacity and Permeability (BCOP) test according to OECD Guideline 437 and EU method B.47 was carried out. Corneas were obtained from fresh isolated bovine eyes of cattle. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. Isolated cornea where observed visually, and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns). Three corneas were used per group (negative control with 0.9% sodium chloride solution, positive control with N,N-dimethylformamide or test item group). The test item preparation, the negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber. 750 µL of either the test item, negative or positive control were administered. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1 °C for 10 minutes. Subsequently, corneal surfaces were washed at least three times with wash medium and finally with incubation medium. Fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± °C.

Opacity was measured by an opacitometer, permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically (OD490, microplate reader). In result, after treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.3 - 5.5). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 100.0 (study acceptance criteria range: 77.1 - 119.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 62.8 and, thus higher than 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1). All validity criteria of the guidelines were fulfilled.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-01-29 to 2018-06-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: reconstructed human cornea-like epithelium

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability:
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.
- Characterisation of the test system:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: Run 1:27029
Run 2: 27040
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test item: 50 µL per tissue
Negative control: 50 µL per tissue
Positive control: 50 µL per tissue

Duration of treatment / exposure:

30 ± 2 minutes

Duration of post- treatment incubation (in vitro):

12 ± 2 minutes incubation and 120 ± 15 minutes at 37 °C

Number of animals or in vitro replicates:

2 (in total 12 tissues)

Details on study design:

- Details of the test procedure used:

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37 °C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37 °C and 5 % CO2 overnight (16 - 24 hours) without medium exchange.

Pre-Treatment:
After the overnight incubation, the tissues were pre-wetted with 20 µL DPBS and incubated at 37 °C and 5 % CO2 for 30 minutes (± 2 minutes).

Exposure and Post-Treatment:
After the 30 minute DPBS pre-treatment, the test item, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37 °C and 5 % CO2 for 30 ± 2 minutes.
At the end of the 30 ± 2 minutes treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (37 °C) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material.
After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 12 ± 2 minutes at room temperature.

After the 12 ± 2 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37 °C) assay medium for 120 ± 15 minutes at 37 °C and 5 % CO2.

- RhCE tissue construct used, including batch number:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: Run 1:27029
Run 2: 27040
Keratinocyte strain: 4F1188
Supplier: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

- Doses of test chemical and control substances used: 50 µL
- Duration and temperature of exposure, post-exposure: exposure: 30 ± 2 min at 37 °C; post-exposure: 12 ± 2 min at room temperature + 120 ± 15 min at 37 °C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.
- Number of tissue replicates used per test chemical and controls: 2
- Wavelength used for quantifying MTT formazan: 570 nm
- Description of the method used to quantify MTT formazan:
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37 °C and 5 % CO2.

The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a pre-labeled 24-well plate containing 2 mL isopropanol so that isopropanol was flowing into the insert on the tissues surface. The plate was sealed with a standard plate sealer. To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
If the test item-treated tissue viability is >60.0 % relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is <60.0 % relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptance Criteria:
The results are acceptable if:
1. The negative control OD >0.8 and <2.5,
2. The mean relative viability of the positive control is:
a) 30 minute exposure: below 50 % of control viability
b) 6 hour exposure: below 50 % of control viability
3. Acceptable variability between tissue replicates: <20 %

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 1 - Tissue 1

Value:

46.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 1 - Tissue 2

Value:

60.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 1 - Mean

Value:

53.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 2 - Tissue 1

Value:

28.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 2 - Tissue 2

Value:

32.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Viability (%)

Run / experiment:

Run 2 - Mean

Value:

30.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct MTT reduction: no
- Colour interfrence with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Results from Run 1

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	1.599	97.7 %	1.675	102.3 %	1.637	100.0%	3.25	4.6 %
Positive Control	0.537	32.8 %	0.618	37.8 %	0.578	35.5 %	3.54	5.0 %
Test item	0.768	46.9 %	0.993	60.7 %	0.881	53.8 %	9.76	13.8 %

 

Table 2: Results from Run 2

Group	Tissue 1		Tissue 2		Mean		SD	Difference between tissue replicates
	OD	Viability	OD	Viability	OD	Viability	Viability
Negative Control	2.199	99.9 %	2.202	100.0 %	2.201	100.0%	0.07	0.1 %
Positive Control	1.156	52.5 %	0.998	45.3 %	1.077	48.9 %	5.09	7.2 %
Test item	0.623	28.3 %	0.714	32.4 %	0.669	30.4 %	2.90	4.1 %

 

 

Interpretation of results:

other: no prediction can be made

Conclusions:

Following treatment with the test item, the tissue viability was 53.8 % in the first run and 30.4 % in the second run and, thus, lower than 60 % in both runs, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model (RhCE) according to OECD 492.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 (± 2) minutes. 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 12 (± 2) minutes at room temperature and further 120 (± 15) minutes with fresh medium at 37 °C.

After treatment with the negative control (sterile deionized water) the mean OD in run 1 was 1.637 (study acceptance criterion: >0.8 and <2.5) and in run 2 was 2.201. Treatment with the positive control (methyl acetate) revealed a mean viability value of 35.3 % in run 1 (study acceptance criterion: <50 %) and 48.9 % in run 2. Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 53.8 % in the first run and 30.4 % in the second run and, thus, lower than 60 % in both runs, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation / corrosion

OECD 431

An in vitro study in accordance with the OECD Guideline OECD 431 and EU Method B.40.bis was performed to assess the skin corrosion potential of the test item. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and 1 hour. The positive control was treated only for 1 hour. 40 ± 3 µL of either the negative control (deionised water), the positive control (potassium hydroxide, 8N) or the test item were applied to the tissues. The test item did not reduce MTT, and it did not indicate colour interference. All of the acceptability criteria were met. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.7 % and, thus, lower than the historically established threshold of 1.01 %. After treatment with the negative control (deionised water) the mean ODs were 1.852 (3 minutes exposure) and 1.578 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.587 and 1.422, respectively.

Following treatment with the test item, the tissue viability was >50 % after 3 minutes exposure (mean viability: 100.8 %) and <15 % after 1 hour exposure (mean viability: 9.8 %), therefore, the test item is considered as corrosive to skin (UN GHS Category 1) (reference 7.3.1-1).

The available in vivo data on skin irritation (publications, harmonised classification) were used to assess the skin irritating potential of the test item in a weight of evidence approach.

The results of the available reports on skin irritation in rabbits show no to light skin irritating potential of the test item (reference 7.3.1-2 to 7.3.1-5). Data in humans did not show a skin irritating potential (reference 7.10.5-1). Additonally, a harmonised classification according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation) for the test item is published. As consequence a worst case scenario is anticipated for classification. Therefore and in accordance with REACH Regulation (EG) No 1907/2006, Annex XI (1), the classification for skin irritation follows the harmonised classification (UN GHS, Category 2, H315) and no further in vivo or in vitro studies were performed.

Eye irritation

OECD 492

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model (RhCE) according to OECD 492 (reference 7.3.2-2).

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential.

Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 30 (± 2) minutes. 50 µL of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. The post-treatment incubation was 12 (± 2) minutes at room temperature and further 120 (± 15) minutes with fresh medium at 37 °C.

After treatment with the negative control (sterile deionized water) the mean OD in run 1 was 1.637 (study acceptance criterion: >0.8 and <2.5) and in run 2 was 2.201. Treatment with the positive control (methyl acetate) revealed a mean viability value of 35.3 % in run 1 (study acceptance criterion: <50 %) and 48.9 % in run 2. Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 53.8 % in the first run and 30.4 % in the second run and, thus, lower than 60 % in both runs, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

OECD 437

To assess the ocular corrosiveness and eye irritating properties a GLP-compliant Bovine Corneal Opacity and Permeability (BCOP) test according to OECD Guideline 437 and EU method B.47 was carried out. Corneas were obtained from fresh isolated bovine eyes of cattle. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. Isolated cornea where observed visually, and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns). Three corneas were used per group (negative control with 0.9% sodium chloride solution, positive control with N,N-dimethylformamide or test item group). The test item preparation, the negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber. 750 µL of either the test item, negative or positive control were administered. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1 °C for 10 minutes. Subsequently, corneal surfaces were washed at least three times with wash medium and finally with incubation medium. Fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± °C.

Opacity was measured by an opacitometer, permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically (OD490, microplate reader). In result, after treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.3 - 5.5). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 100.0 (study acceptance criteria range: 77.1 - 119.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 62.8 and, thus higher than 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1). All validity criteria of the guidelines were fulfilled (reference 7.3.2-1).

However, a harmonised classification according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation) is available. Therefore, the test item will be classified as eye irritant (Category 2, H319).

Justification for classification or non-classification

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

The available data for skin irritation/corrosion are sufficient for classification purposes under Regulation (EC) No 1272/2008. As a result the test item is considered to be classified for skin corrosion (UN GHS, Category 1, H314) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

The available data for eye irritation are sufficient for classification purposes under Regulation (EC) No 1272/2008. As a result the test item is considered to be classified for eye irritation (UN GHS, Category 1, H318) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.

However, a harmonised classification for eye irritation according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation) is available. Therefore, the test item is considered to be classified as eye irritant (Category 2, H319) according to Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.