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Endpoint summary

Description of key information

Skin and eye irritation potential of the test substance.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 09 November 2017 to 12 January 2018. Report issue: 21 August 2018.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Multiple adult human donors

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SYSTEM
- Source: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France). EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 09 January 2018. On the day of experiment EpiSkin™ tissues were transferred to 12-well plates with maintenance medium / the pre-incubation phase of the EpiSkin™ tissues started.

TEMPERATURE AND OTHER TEST CONDITIONS
- Pre-warming: Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium. The EpiSkin™ tissues were incubated for 22.5 hours.

- Temperature and other test conditions used during treatment / exposure: 37 ± 1.5 °C, 5 ± 0.5% CO2 for 15 minutes
- Temperature and other test conditions post-treatment incubation: 37 ± 1.5 °C, 5 ± 0.5% CO2 for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Once - the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.


MTT ASSAY
- Method: A 12-well plate was filled with 2 mL assay medium containing 0.3 mg/mL MTT per well. After the incubation period was completed for all tissues the cell culture inserts were transferred from the holding plates to the MTT-plate. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was rinsed three times with PBS and the tissues were plotted. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol containing 0.04 N HCl) into each vial. The tissue samples were completely covered by isopropanol. The formazan salt was extracted for about 2.5 hours at room temperature with gentle agitation. For each tissue sample 2 x 200 µL aliquots of the formazan extract were transferred into a 96-well flat bottom microtiter plate for optical density reading..
- Spectrophotometer: Versamax (Molecualr Devices, 85737 Isaminng, Germany)
- Wavelength: 570 nm

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: Three tissue samples per endpoint

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
10 µL (26.3 µL/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight):
10 µL


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

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After the end of the treatment interval the inserts were immediately removed from the 12-well plate. The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5% CO2.

Number of replicates:

Negative control = 3
Negative Control Freeze Killed = 3
Positive control = 3
Test Item = 3
Test Item Freeze Killed Tissue = 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 1

Value:

70.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 2

Value:

62.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item Tissue No 3

Value:

55.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean (of the 3 tissue samples tested)

Value:

60.13

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item passed the colour interference pre-test.

The test item reduced MTT in the pre-test for direct MTT reduction. Consequently, additional tests with freeze-killed tissues were necessary to show the amount of MTT reduction due to residual NADH and associated enzymes within the killed tissue. The relative viabilities were correct for MTT reduction in killed tissue.

Data Evaluation

For the test item and the positive control, the mean relative viability ±rel. standard deviation of the three individual tissues was calculated and used for classification according to the following prediction model:

In Vitro Result	in vivo Prediction	EU Classification	UN GHS Classifciation
mean tissue viability <50%	Irritant	H315	Category 2
mean tissue Viability > 50%	Non-irritant

 

For the current test, an irritation potential leading to H315 classification of EU according to regulation (EC) 1272/2008, and GHS category 2 according to UN GHS (published 2003, last (7^th revision 2017) is recommended if the mean relative tissue viability of three individual tissues is reduced to<50% of the negative control. After treatment with the test item the mean relative absorbance value was reduced to 60.13%.This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of the test, the test substance is not irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

Introduction

The in vitro skin irritation potential of the test substance as assessed a Human Skin Model Test design to meet the following guideline:

• OECD Guideline for the testing of Chemicals 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (Original Guideline adopted July 28, 2015)

Method

Three tissues of the human skin model EpiSkin™ were treated with the test item, a negative control (PBS) or a positive control (5% sodium lauryl sulfate) for 15 minutes at 37 ± 1.5 °C, 5 ± 0.5% CO2. Tissues were then washed to remove test material and following a further 42 hours post exposure incubation an MTT assay was run on the tissue samples to assess tissue viability were included.

The test item reduced MTT (pre-test for direct MTT reduction); however, it did not dye water, when mixed with it (pre-test for colour interference). Due to MTT reduction additional tests with freeze-killed tissues were necessary.

Result

The negative and positive control absorbance values were well within the required acceptability criterion thus showing the quality and validity of the test system.

Following treatment with the test item the mean relative absorbance value decreased to 60.13%. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

Conclusion

In conclusion, it can be stated that in this study and under the experimental conditions reported the test substance is not irritant to skin according to UN GHS and the EU CLP regulation.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not 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:

key study

Study period:

Experimental phase: 24 November 2017. Report issues: 17 April 2018.

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

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

Collection of Bovine Eyes
Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularisation, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of either test substance, negative control or positive control covering the anterior compartment of the eye.

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Two hours

Number of animals or in vitro replicates:

Three replicates (cornea) per treatment (test substance, negative control, positive control)

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS : Yes

NUMBER OF REPLICATES : three per treatment

NEGATIVE CONTROL USED: Yes, Saline (0.9% NaCl in deionised water)

SOLVENT CONTROL USED: No

POSITIVE CONTROL USED : Yes, 2-Ethoxyethanol (purity: 99%)

APPLICATION DOSE AND EXPOSURE TIME : 10 minutes

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After expsoure, the test and control substances were rinsed off from the application side with saline, fresh cMEM was then added into the anterior compartment.

POST-INCUBATION PERIOD: Yes, two-hours

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom France))
- Corneal permeability: passage of sodium fluorescein dye measured with microtitre plate reader at 490nm.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Irritation parameter:

in vitro irritation score

Run / experiment:

Test substance Exposure Cornea 1

Value:

21.19

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance Exposure Cornea 2

Value:

11.98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Test Substance Exposure Cornea 3

Value:

14.49

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean IVIS Score for Test Substance

Value:

15.89

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Relative to the negative control, the test substance caused an increased in corneal opacity. The calculated mean IVIS was 15.89 which is below the threshold for serious eye damage (IVIS > 55).

Test Results (10 Minute Treatment time)

Test Group	Opacity Value = Difference (t130 -t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	In vitro Irritancy Score
		Mean		Mean
Negative Control	1	0.67	0.061	0.078	1.92	1.86	No category
	1		0.068		2.02
	0		0.109		1.64
Positve Control	56.33*		1.703*		81.87	87.23	Category 1
	61.33*		1.652*		86.11
	73.33*		1.359*		93.71
Test Substance	21.33*		-0.009*		21.19	15.89	No prediction can be made
	12.33*		-0.023		11.98
	14.33*		0.011		14.49

*corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the experimental conditions reported, the test substance does not cause serious eye damaging (i.e. it is not classified GHS Category 1 for eye effects).

Executive summary:

Introduction

This in vitro study was performed to assess the corneal damage potential of the test substance by means of the BCOP assay using fresh bovine corneae. The test was designed to meet the requirements of OECD Guideline for Testing of Chemicals 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 (July, 2013).

Method

After a first opacity measurement of the fresh bovine corneae (t₀), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contain incubation medium as well. Afterwards,opacity was measured a second time (t₁₃₀).

After the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.

Results

With the negative control (0.9% (w/v) NaCl solution in deionised water) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability ofthe corneae corresponding to a classification as serious eye damaging (CLP/EPA/GHS(Cat 1)).

 

Relative to the negative control, the test substance caused an increase of the corneal opacity. The calculated mean in vitro irritancy score was 15.89 which is below the threshold for classification for serious eye damage (IVIS > 55) according to CLP/EPA/GHS.

 

Conclusion

In conclusion, under the experimental conditions reported, the test substance does not cause serious eye damaging (i.e. it is not classified CLP/EPA/GHS Category 1 for eye effects).

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 23 July 2018 to 21 August 2018. Report issued: 19 October 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

Due to a technician error, the eight-hour post-dose analgesia for the second treated animal was not performed. This deviation was considered to have not affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Two New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK.
- Age at study initiation: 12 to 52 weeks old.
- Weight at study initiation: 3.192 to 3.158g
- Housing: Individually housed in suspended cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

Two rabbits

Details on study design:

Study Design
Only animals free of ocular damage were used.

Analgesia
A subcutaneous injection of buprenorphine 0.01 mg/kg was administered 60 minutes prior to test item application to provide a therapeutic level of systemic analgesia. Five minutes prior to test item application, a pre-dose anesthesia of ocular anesthetic (two drops of 0.5% proxymetacaine hydrochloride) was applied to each eye.

Initially, a single rabbit was treated. A volume of 0.1 mL of the test item was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Eight hours after test item application, a subcutaneous injection of post-dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

After consideration of the ocular responses produced in the first treated animal, a second animal was similarly treated.

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment, according to the numerical evaluation (Draize, J.H, 1977).

Any other ocular effects were also noted. Examination of the eye was facilitated by the use of the light source from a standard ophthalmoscope.

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

6

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

6

Max. score:

2

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

3

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks:

All eyes of study animals appeared normal after 14 days.

Other effects:

One animal showed a slight body weight loss during the study.

Scattered or diffuse corneal opacity was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations.

Iridial inflammation was noted in both treated eyes one hour after treatment and at the 24 and 48-Hour observations.

Moderate conjunctival irritation was noted in both treated eyes one hour after treatment and at the 24, 48 and 72-Hour observations. Minimal conjunctival irritation was noted in both treated eyes at the 7-Day observation.

Both treated eyes appeared normal at the 14-Day observation.

Interpretation of results:

Category 2A (irritating to eyes) based on GHS criteria

Conclusions:

The test item produced a maximum group mean score of 26.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item was classified as Category 2A (irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required.

Executive summary:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. The method used was designed to meet the requirements of the following guidelines:

• OECD Guideline for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 09 October 2017)
• Method B5 Acute Toxicity (Eye Irritation) of Commission Regulation (EC) No. 440/2008

Results

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation, iridial inflammation and scattered or diffuse corneal opacity. Both treated eyes appeared normal at the 14 day observation.

Conclusion

The test item produced a maximum group mean score of 26.5 and was classified as a moderate irritant (Class 5 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

The test item was classified as Category 2A (irritating to eyes) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item was also classified as Irritating to eyes (Category 2) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. The Signal Word “Warning” and the Hazard Statement “H319: Causes serious eye irritation” are therefore required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available