Phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2'-iminobis[ethanol]

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 12, 2016 to September 19, 2016

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:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

but the study integrity was not adversely affected by the deviations

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: RE 10-7
Purity/composition: 10% dry matter
Appearance: withish liquid

Test system:

human skin model

Remarks:

human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM))

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The test is based on the experience that irritant chemicals show cytotoxic effects following short term exposure to the stratum corneum of the epidermis. The test was designed to predict and classify the skin irritation potential of a test substance by assessment of its effect on a three dimensional human epidermis model. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

The test substance was applied undiluted (25 µl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 µL

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:

Reduction of MTT to MTT formazan

Value:

78

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:

Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test substance compared to the negative control tissues was 78%. Since it was above 50% after 15 ± 0.5 minutes treatment the test substance was considered to be non-irritant. The positive control had a mean cell viability of 15% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the reference items was less than 13%, indicating that the test system functioned properly.
It was concluded that this test was valid and that the test substance was non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the substance was considered to be non-irritant to human skin.

Executive summary:

A study was conducted to determine the in vitro skin irritation potential of the substance according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Human three dimensional epidermal tissue model was exposed in triplicate to at least 25 µL test substance for 15 min. After a 42 h post-incubation period, a determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test substance compared to the negative control tissues was 78%. Since it was above 50% the test substance was considered to be non-irritant. The positive control showed a mean cell viability of 15% after 15 ± 0.5 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the reference substance was less than 13%, indicating that the test system functioned properly. Under the study conditions, the substance was considered to be non-irritant to human skin (Verbaan, 2016).

Reference 2

Endpoint:

skin irritation: in vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reason / purpose for cross-reference:

data waiving: supporting information

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

Remarks:

Bovine Corneal Opacity and Permeability (BCOP) test

Type of information:

experimental study

Adequacy of study:

key study

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:

yes

Remarks:

but the study integrity was not affected by this deviation

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: RE 10-7
Purity/composition: 10% dry matter
Appearance: whitish liquid

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µL

Duration of treatment / exposure:

10 min

Duration of post- treatment incubation (in vitro):

After exposure the cornea was thoroughly rinsed to remove the test substance and incubated for 2 hours with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 minutes incubation period with sodium fluorescein.

Number of animals or in vitro replicates:

3

Details on study design:

The Bovine Corneal Opacity and Permeability (BCOP) test is an organic model that provides short-term maintenance of normal physiological and biological function of the bovine cornea in an isolated system. In this test method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability with an opacitymeter and an ultraviolet/visible spectrophotometer, respectively.

Irritation parameter:

in vitro irritation score

Run / experiment:

corneal opacity and permeability

Value:

34

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no prediction could be made

Other effects / acceptance of results:

The test substance was applied as it is (750 µL) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 51 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Since the test substance induced an IVIS > 3 ≤ 55, no prediction can be made.

Interpretation of the results:

- In vitro irritancy score

The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: in vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:

< or = 3: no category,

>3 and < or = to 55: no prediction can be made,

> 55: category 1.

Conclusions:

Under the study conditions, no prediction for eye irritation could be made as the substance induced an in vitro irritancy score of 34 (IVIS > 3 ≤ 55).

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the substance according to OECD Guideline 437, in compliance with GLP. Freshly collected bovine corneas were exposed to 750 µL test substance for 10 min. After exposure, the corneas were thoroughly rinsed to remove the test substance and incubated for 2 h with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 min incubation period with sodium fluorescein. An in vitro irritancy score was then established. Physiological saline and ethanol were used as negative and positive controls, respectively. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 51 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the study conditions, no prediction for eye irritation could be made as the substance induced an in vitro irritancy score of 34 (IVIS > 3 ≤ 55) (Verbaan, 2016).  

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

EpiOcular™ Cornea Epithelial Model

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 28 2016 to December 02, 2016

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

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity/composition: 10.0% dry matter
Appearance: whitish liquid

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test substance to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL

Duration of treatment / exposure:

30 min

Observation period (in vivo):

-

Duration of post- treatment incubation (in vitro):

After exposure the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 h at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect.

Number of animals or in vitro replicates:

3

Details on study design:

The test consists of application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 min. After exposure the cornea epithelial construct is thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 hours incubation period determination of the cytotoxic (irritancy) effect is performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement - optical density reading at 570 nm). Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance.

Irritation parameter:

other: cell viability (%)

Run / experiment:

reduction of MTT

Value:

3.1

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:

The test substance showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test substance was 0.51% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 3.1%. Since the mean relative tissue viability for the test substance was below or equal to 60% after 30 ± 2 min treatment, it was considered to be potentially irritant or corrosive to the eye.
The positive control had a mean cell viability of 27% after 30 ± 2 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 1.5, which is within the acceptability range from > 0.8 to < 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 4%, indicating that the test system functioned properly. It was concluded that this test was valid and that the test substance was potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the study conditions, the substance was considered to be irritant to human eyes.

Executive summary:

A study was conducted to determine the in vitro eye irritation potential of the substance according to OECD Guideline 492, in compliance with GLP. The EpiOcular tissue construct is a non-keratinized epithelium prepared from normal human keratinocytes. The test consisted of an application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 min. After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 h incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3 -(4,5 -dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement by optical density reading at 570 nm).  Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The test substance showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test substance was 0.51% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 3.1% which was below 60%. Therefore, the test substance was considered to be irritant or corrosive to the eyes. The positive control showed a mean cell viability of 27% after 30 ± 2 min exposure. The absolute mean OD570 of the negative control tissues was 1.5, which is within the acceptability range of > 0.8 to < 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 4%, indicating that the test system functioned properly. Under the study conditions, the substance was considered to be irritant to human eyes (Verbaan, 2017).

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 06, 2017 to April 11, 2017

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch no.: RE 10-9
Purity/composition: 92.75% dry matter
Appearance: lightly yellow paste

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Species: Rabbit
Strain: New Zealand White
Condition: SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany
Number of Animals: 3 Males
Age at the Initiation of Dosing: Young adult animals (approximately 28-30 weeks old) were selected
Weight at the Initiation of Dosing: 3963 to 4146g
The actual daily mean temperature during the study period was 20°C with an actual daily mean relative humidity of 58 to 63%. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.
Feed: Pelleted diet for rabbits (Global Diet 2030 Teklad®, Mucedola, Milanese, Italy), ad libitum
Water: Municipal tap-water was freely available to each animal via water bottles

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

0.1 mL in the conjunctival sac of one of the eyes after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of the test substance.

Duration of treatment / exposure:

Immediately after the 24-hour observation, a solution of 2% fluorescein in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. This procedure was repeated to assess recovery after 72 hours and 7 and 14 days after treatement. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area.

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

3

Details on study design:

Single samples of 0.1 mL of phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2’-iminobis[ethanol] were instilled into one eye of each of three rabbits. Observations were made 1, 24, 48 and 72 hours and 7, 14 and/or 21 days after instillation.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0.7

Max. score:

1

Reversibility:

fully reversible within: 72 fours

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

mean

Time point:

24/48/72 h

Score:

2.8

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

conjunctivae score

Remarks:

chemosis

Basis:

mean

Time point:

24/48/72 h

Score:

1.8

Max. score:

3

Reversibility:

fully reversible within: 14 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 14 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

48 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

72 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 21 d

Irritant / corrosive response data:

Instillation of approximately 0.1 mL of the test substance into one eye of each of three rabbits resulted in effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent 7 and/or 14 days after instillation. The corneal injury resolved within 14 days. Iridial irritation was observed in all animals and resolved within 72 hours. The irritation of the conjunctivae consisted of redness, chemosis and discharge and completely resolved within 14 days.
Based on these results:
• according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2’-iminobis[ethanol] should be classified as : irritating to eyes (Category 2A).
• according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), phosphoric acid, mono- and di-isotridecyl esters, compd. with 2,2’-iminobis[ethanol] should be classified as Irritating to eyes (Category 2) and labeled as H319: Causes serious eye irritation.

Other effects:

- No signs of systemic toxicity were observed in the animals during the test period and no mortality occurred.
- Remnants of the test substance were present on the outside of the eyelids on Day 1 for one animal only. This is considered to be due to innate protective physiological mechanisms of the eye against entry of foreign compounds.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the study conditions, the substance was considered to be irritating to rabbit eyes.

Executive summary:

A study was conducted to determine the in vivo eye irritation potential of the substance according to OECD Guideline 405, EU Method B.5 and EPA OPPTS 870.2400, in compliance with GLP. 0.1 mL of the test substance were instilled into one eye of three male New-Zealand rabbits. Observations (corneal opacity, iris irritation and conjunctivae redness and chemosis) were made 1, 24, 48 and 72 h as well as 7 and 14 d after instillation. Instillation of the undiluted test substance resulted in adverse effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent until 14 d after instillation. Iridial irritation was observed in all animals. The irritation of the conjunctivae consisted of redness, chemosis and discharge. All adverse effects were completely resolved within 21 d. Under the study conditions, the substance was considered to be irritating to rabbit eyes (van Sas, 2017).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro skin irritation study:

A study was conducted to determine the in vitro skin irritation potential of the substance according to OECD Guideline 439 and EU Method B.46, in compliance with GLP. Human three dimensional epidermal tissue model was exposed in triplicate to at least 25 µL test substance for 15 min. After a 42 h post-incubation period, a determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation was expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test substance compared to the negative control tissues was 78%. Since it was above 50% the test substance was considered to be non-irritant. The positive control showed a mean cell viability of 15% after 15 ± 0.5 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically with the reference substance was less than 13%, indicating that the test system functioned properly. Under the study conditions, the substance was considered to be non-irritant to human skin (Verbaan, 2016).

In vitro eye irritation studies:

A study was conducted to determine the in vitro eye irritation potential of the substance according to OECD Guideline 437, in compliance with GLP. Freshly collected bovine corneas were exposed to 750 µL test substance for 10 min. After exposure, the corneas were thoroughly rinsed to remove the test substance and incubated for 2 h with fresh medium followed by opacity measurement and the permeability of the corneas was determined after a 90 min incubation period with sodium fluorescein. An in vitro irritancy score was then established. Physiological saline and ethanol were used as negative and positive controls, respectively. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (ethanol) was 51 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the study conditions, no prediction for eye irritation could be made as the substance induced an in vitro irritancy score of 34 (IVIS > 3 ≤ 55) (Verbaan, 2016).  

A study was conducted to determine the in vitro eye irritation potential of the substance according to OECD Guideline 492, in compliance with GLP. The EpiOcular tissue construct is a non-keratinized epithelium prepared from normal human keratinocytes. The test consisted of an application of the test substance (50 µL) to the surface of the cornea epithelial construct for 30 min. After exposure, the cornea epithelial construct was thoroughly rinsed to remove the test substance and transferred to fresh medium for an immersion incubation. After transfer to fresh medium for a 2 h incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at the end of the treatment (mean absorption measurement by optical density reading at 570 nm).  Eye hazard potential is expressed as the remaining cell viability after exposure to the test substance. The test substance showed colour interference in aqueous conditions. In addition to the normal procedure, two tissues were treated with test substance. Instead of MTT solution these tissues were incubated with assay medium. The non-specific colour of the test substance was 0.51% of the negative control tissues. The OD of the treated tissues without MTT assay was subtracted from the ODs of the test substance treated viable tissues with MTT assay. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test substance compared to the negative control tissues was 3.1% which was below 60%. Therefore, the test substance was considered to be irritant or corrosive to the eyes. The positive control showed a mean cell viability of 27% after 30 ± 2 min exposure. The absolute mean OD570 of the negative control tissues was 1.5, which is within the acceptability range of > 0.8 to < 2.5. The standard deviation value of the percentage viability of two tissues treated identically was less than 4%, indicating that the test system functioned properly. Under the study conditions, the substance was considered to be irritant to human eyes (Verbaan, 2017).

In vivo eye irritation study:

A study was conducted to determine the in vivo eye irritation potential of the substance according to OECD Guideline 405, EU Method B.5 and EPA OPPTS 870.2400, in compliance with GLP. 0.1 mL of the test substance were instilled into one eye of three male New-Zealand rabbits. Observations (corneal opacity, iris irritation and conjunctivae redness and chemosis) were made 1, 24, 48 and 72 h as well as 7 and 14 d after instillation. Instillation of the undiluted test substance resulted in adverse effects on the cornea, iris and conjunctivae. The corneal injury consisted of opacity and epithelial damage. As a result of the corneal injury, pannus (neovascularization of the cornea) was apparent until 14 d after instillation. Iridial irritation was observed in all animals. The irritation of the conjunctivae consisted of redness, chemosis and discharge. All adverse effects were completely resolved within 21 d. Under the study conditions, the substance was considered to be irritating to rabbit eyes (van Sas, 2017).

Justification for classification or non-classification

Skin irritation:

Based on the results of anin vitroskin irritation study, the test substance does not need to be classified according to EU CLP (EC 1272/2008) criteria.

Eye irritation:

Based on the results inin vitroandin vivoeye irritation studies the substance is considered to be irritating to eyes in. Hence, it warrants a classification of Eye Irrit. 2 - H319 (causes serious eye irritation) according to EU CLP (EC 1272/2008) criteria.