Reaction mass of lauric acid, compound with morpholine (1:1) and 2-ethylhexyl dihydrogen phosphate, compound with morpholine (1:2) and bis(2-ethylhexyl) hydrogen phosphate, compound with morpholine (1:1)

Endpoint summary

Description of key information

Based on data available the test item is considered to have irritating, but non-corrosive properties to skin.

Available in vitro data on eye corrosive / severe irritating properties, indicate non-corrosive properties of the test item. Data obtained from an in vivo study indicate eye irritating potential.

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:

2016-04-27 to 2016-06-16

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No 440/2008, Annex Part B, B.40Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142, dated May 31st, 2008.

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol No. 118; “EPISKINTM Skin Corrosivity Test” updated December 2011 / February 2012 (ECVAM Database Service on Alternative Methods to Animal Experimentation).

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EPISKIN model has been validated for corrosivity testing in an international trial; it is considered to be suitable for this study (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 16-EKIN-018
- Expiry date: 09 May 2016
- Date of initiation of testing: 04 May 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: approx. 25 µL, one time
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL pe rwell
- Incubation time: 3 h
- Spectrophotometer: not specified
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. In addition, the test item showed no ability to become coloured in contact with water. Thus, additional controls and data calculations were not necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin (Cat. 1A) if the viability after 3 minutes exposure is less than 35%.
- The test substance is considered to be corrosive to skin (Cat 1B and 1C) if the viability after 3 minutes exposure is greater than or equal to 35% AND after 60 minutes exposure smaller than 35%; or if the viability after 60 minutes exposure is greater than or equal to 35% AND after 240 minutes exposure smaller than 35%.
- The test substance is considered to be non-corrosive to skin if the viability after 4 hours exposure is greater than or equal to 35%.
- Justification for the selection of the cut-off point: The cut-off value of 35 % and classification method was validated in an international validation of this kit (Fentem, 1998). The prediction model corresponds to the methods agreed by EU regulatory agencies in line with OECD 431 (OECD, 2015).

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
- Concentration: 9 g/L

POSITIVE CONTRORL
- Amount applied: 50 µL

Duration of treatment / exposure:

4 h

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: According to the test results of in vitro skin irritation study: No colour change was observed after three hours of incubation during the check-test for possible direct MTT reduction with test item. The test item did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. Furthermore, the test item was completely removed from the epidermal surface at rinsing period. Additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the two negative control tissues was 1.455.
- Acceptance criteria met for positive control: yes. The positive control result showed 2 % viability. The difference of viability between the two tissue replicates:
Negative control: 0 %
Positive control: 1 %
Test item: 0 %
- Acceptance criteria met for variability between replicate measurements: All validity criteria were within acceptable limits in the experiment therefore the study can be considered as valid.

Interpretation of results:

other: not classified as corrosive

Conclusions:

In conclusion, in this in vitro skin corrosion test using the EPISKIN model with the test item the results indicated that the test item is not corrosive to skin. In conclusion, according to the UN GHS classification the test item can be classified as Non-corrosive, under the utilised testing conditions. However, further data is required in order to predict skin irritating properties.

Executive summary:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1 x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 100 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-02-23 to 2016-04-06

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:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

other: reconstructed human epidermis

Cell type:

non-transformed keratinocytes

Cell source:

other: adult donors

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTMSM, EPISKIN SNC Lyon, France
- Tissue batch number: 16-EKIN-008
- Expiry date: 29 February 2016
- Date of initiation of testing: 24 February 2016

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: After the incubation time, the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1 x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source (care was taken to avoid damaging to the epidermis).
- Observable damage in the tissue due to washing: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 h
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean OD value negative control: 0.826;
Historical Mean OD value negative control (Period of 2011 to 2016-02)): 0.800
- Barrier function: IC50 = 2.0 mg/mL (18 h treatment with SDS 1.5 mg/mL)
- Morphology: Well differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: EpiSkinTMSM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
- Reproducibility: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item showed no direct interaction with MTT. Using of additional control was not necessary.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the negative control.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 hours post incubation is greater than 50% of the negative control.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 10 µL

NEGATIVE CONTROL
- Amount applied: 10 µL
- Concentration: 1 x PBS

POSITIVE CONTROL
- Amount applied: 10 µL
- Concentration: 5 % aq. solution

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

27

Vehicle controls validity:

not applicable

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 change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be excluded.
- Colour interference with MTT: As the test item has an intrinsic colour (yellowish), two additional test item-treated tissues were used for the non-specific OD evaluation. The mean OD (measured at 570 nm) of these tissues was determined as 0.016. The Non Specific Colour % (NSC %) was calculated as 1.9 %. Therefore additional data calculation was not necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT. demonstrated the technical proficiency in a separate study (study no.: 392.554.2938) using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD value of the three negative control tissues was 0.826.
- Acceptance criteria met for positive control: yes. The mean OD value obtained for the positive control was 0.031 and this result corresponds to 4 % viability when compared to the results obtained from the negative controls.
- Acceptance criteria met for variability between replicate measurements: Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Interpretation of results:

other: the test item is Irritant and/or Corrosive

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, with the test item indicated that the test item is Irritant (UN GHS Category 2) and/or Corrosive (UN GHS Category 1). However, this test method (OECD 439) cannot resolve between UN GHS Categories 1 and 2. Therefore further information on skin corrosion will be required to decide on its final classification.

Executive summary:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. As the test item has an intrinsic colour (yellowish), two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. The test item showed significantly reduced cell viability in comparison to the negative control (mean value: 27 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

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:

2016-05-06 to 2016-06-16

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:

2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (19.2ºC to 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
- Time interval prior to initiating testing: approx. 2 h
- indication of any existing defects or lesions in ocular tissue samples: none

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: approx. 30 µL

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 5 drops/minute. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and, after being placed in the superfusion apparatus, the eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness, opacity, and fluorescein retention to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ±5-7 % within approximately 45 to 60 minutes before the start of application. No changes in thickness were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl (9 g/L) Saline

POSITIVE CONTROL USED: Acetic acid 10% (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 30 µL, 10 sec

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

METHODS FOR MEASURED ENDPOINTS:
- Damage to epithelium based on fluorescein retention: The fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5).
- Swelling: The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm.

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment
- morphological effects (e.g., pitting or loosening of the epithelium)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Irritation parameter:

percent corneal swelling

Run / experiment:

At up to 75 min

Value:

7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

percent corneal swelling

Run / experiment:

At up to 240 min

Value:

18

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: yes. Based on the overall ICE Class the positive control Acetic acid 10 % (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
Positive and negative control values were within the corresponding historical control data ranges.

Interpretation of results:

other: non-corrosive

Conclusions:

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 1xII, 1xIII, 1xIV. According to the guideline OECD 438, the test item is categorized as “No prediction can be made”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (~30 μL /eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. Test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Reference 2

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-06-20 to 2016-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: S & K LAP Kft.
- Age at study initiation: Young adult rabbits, 11 weeks old
- Weight at study initiation: 2908-3102 g
- Housing: Animals were housed individually in metal cages
- Diet: C.HYF rabbit mixed diet produced by Cargill Takarmány Zrt., 5300 Karcag, Madarasi út 0399., Hungary, ad libitum
- Water: tap water from watering bottles ad libitum
- Acclimation period: 5 days in first animal, 6 days in second animal and 7 days in third animal

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): above 10
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL

Duration of treatment / exposure:

24 h

Observation period (in vivo):

1, 24, 48 and 72 hours then 1 and 2 weeks after the application. Any clinical signs of toxicity or signs of pain and/or distress were recorded twice daily, with a minimum of 6 hours between observations.

Number of animals or in vitro replicates:

3

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The eyes of the test animals were not washed out 24 hours after the application of test item, because the test substance was removed from the eye of the test animal by physiological mechanisms and the test item did not cause immediately severe irritation or corrosion after test item application.

SCORING SYSTEM: scoring system according to Draize (1979) and OECD 405 (02 October 2012)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.66

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

1.66

Max. score:

3

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 2 weeks

Other effects:

- Lesions and clinical observations: No systemic toxicity was observed.
- Effects of rinsing or washing: no washing performed
- Other observations: Sign of pain and distress as discharge was observed in all animals between the treatment day and Day 4.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, the test item applied to the rabbits’ eye mucosa, caused slight to severe conjunctival and slight to moderate cornea irritant effects which were fully reversible within two weeks.

Executive summary:

The acute eye irritation study of the test item was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012). The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A volume of 0.1 mL of the test item solution was used, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours, the 1 and 2 weeks after the application. One hour after the treatment, slight conjunctival redness, slight to severe chemosis, severe discharge, as well as slight to moderate cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area. One week after the treatment, slight conjunctival redness and cornea opacity were recorded. The area of cornea involved was greater than three quarters, up to the whole area. Two weeks after the treatment all animals were free of symptoms and the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows: cornea opacity: 1.66, 1.33, 2.00 iris: 0.00, 0.00, 0.00 redness: 1.00, 1.66, 1.66 chemosis: 2.00, 2.00, 2.00 discharge: 2.66, 3.00, 3.00 No systemic toxicity was observed on the day of the treatment and during the 2-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in all animals between the treatment day and Day 4.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

The purpose of this study was to determine the skin irritation potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of epidermal units (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing the epidermal units with 1x PBS solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 and protected from light. The resulting formazan chrystals were extracted with acidified isopropanol and quantified with the optical densities (OD) recorded spectrophotometrically. As the test item has an intrinsic colour (yellowish), two additional test item treated tissues were used for the non-specific OD evaluation. SDS 5 % aq. and 1 x PBS treated (three units / positive and negative control) epidermis units were used as positive and negative controls, respectively. For each treated tissue, viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % when compared to the viability values obtained from the negative control. The test item showed significantly reduced cell viability in comparison to the negative control (mean value: 27 %). All obtained test item viability results were below 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Skin corrosion:

The purpose of this study was to determine the skin corrosion potential of the test item on reconstituted human epidermis in the EPISKIN model in vitro. Disks of EPISKIN (two units) were treated with test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with PBS 1 x solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue, viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control. The test item did not show significantly reduced cell viability in comparison to the negative control after four hours of exposure. Both individual tissue viabilities were above 35 % of the mean negative control value. The average test item treated tissue viability was 100 %. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.

Eye Corrosion / severe Irritant:

Eye Corrosive / severe Irritant in vitro:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (~30 μL /eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" [category 1 of the Globally Harmonised System for the Classification and Labelling of chemicals (GHS)], or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes. Test item, Acetic acid 10 % (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in such a way that the entire surface of the cornea was uniformly covered with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. Positive and negative controls showed the expected results. The experiments were considered to be valid.

Eye Corrosive / severe Irritant in vivo:

The acute eye irritation study of the test item was performed in three New Zealand White rabbits. The irritation effect of the test item was evaluated according to the Draize method (OECD No.: 405, 2012). The test item was placed into the conjunctival sac of the left eye of each animal. The untreated right eye served as control. A volume of 0.1 mL of the test item solution was used, as a single dose. The eyes of the test animals were not washed out after the application of test item. The eyes were examined at 1, 24, 48 and 72 hours, the 1 and 2 weeks after the application. One hour after the treatment, slight conjunctival redness, slight to severe chemosis, severe discharge, as well as slight to moderate cornea opacity were observed. The area of cornea involved was greater than three quarters, up to the whole area. One week after the treatment, slight conjunctival redness and cornea opacity were recorded. The area of cornea involved was greater than three quarters, up to the whole area. Two weeks after the treatment all animals were free of symptoms and the study was terminated. The animals’ individual mean scores (considering readings at 24, 48 and 72 hours after the treatment) were as follows: cornea opacity : 1.66, 1.33, 2.00; iris: 0.00, 0.00, 0.00; redness: 1.00, 1.66, 1.66; chemosis: 2.00, 2.00, 2.00; discharge: 2.66, 3.00, 3.00. No systemic toxicity was observed on the day of the treatment and during the 2-week observation period. The body weight of animals corresponded to their species and age. Sign of pain and distress as discharge was observed in all animals between the treatment day and Day 4. In conclusion, the test item, applied to the rabbits’ eye mucosa, caused slight to severe conjunctival and slight to moderate cornea irritant effects which were fully reversible within two weeks.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on corrosive / irritant properties, the test item is classified and labelled as skin irritant category 2 (H315: "Causes skin irritation") and eye irritant category 2 (H319: "Causes serious eye irritation") according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.