Imidazolium compounds, 2-(C9-19 and C9-19-unsatd. alkyl)-1-[(C10-20 and C10-20-unsatd. amido)ethyl]-4,5-dihydro-1-Me, Me sulfates

Endpoint summary

Description of key information

Based on the results of the in vivo/in vitro skin and eye read across studies, together with the in vitro eye irritation study with substance itself, the test substance, di-C16 and C18-unsatd. AAEMIM-MS, is considered to be irritating to both skin and eyes (in worst case).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From October 31, 2007 to November 22, 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 12 weeks
- Housing: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (Harlan Laboratories Ltd., Füllinsdorf) and haysticks 4642 (batch no. 08/07, Provimi Kliba AG / Switzerland) were provided for gnawing.
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 rabbit maintenance diet (batch no. 52/07) provided by Provimi Kliba AG, 4303 Kaiseraugst /Switzerland.
- Water: ad libitum, municipal water
- Acclimation period: 4-6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23°C
- Humidity (%): 30-70%
- Air changes (per hr): 10-15
- Photoperiod: 12 h light/dark cycle

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

water

Controls:

not specified

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.5 mL test substance was weighed as delivered by the Sponsor and then moistened with approximately 0.5 mL of purified water before application.
- pH: 6.44 (1% solution)

Duration of treatment / exposure:

4 h

Observation period:

14 d

Number of animals:

3 animals (1 male and 2 females)

Details on study design:

PRE-EXPERIMENTAL PROCEDURE:
4 d before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm2 (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was clipped again.

ADMINISTRATION OF THE TEST SUBSTANCE:
On the day of treatment, 0.5 mL of test substance was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape. The application sites were observed for erythema, edema and other dermal findings approximately 1, 24, 48 and 72 hours after patch removal and on study days 7, 10 and 14.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 4 hours
Then the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.

SCORING SYSTEM: The skin reaction was assessed according to the numerical scoring system listed in the Commission Directive 2004/73/EC, April 29, 2004.

Grading of skin reaction

Erythema and Eschar formation
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) or eschar formation
(injuries in depth preventing erythema reading) 4

Oedema formation
No oedema 0
Vera slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (edges raised approximately 1mm) 3
Severe oedema (raised more then 1mm and
extending beyond the are of exposure) 4

Mortality / Viability: the rabbits were observed daily from acclimatization of the animals to the termination of test.

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

not fully reversible within: 14 d

Remarks on result:

other: Scaling present

Irritation parameter:

erythema score

Basis:

animal: #2, #3

Time point:

24/48/72 h

Score:

3

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Scaling present

Irritation parameter:

edema score

Basis:

animal: #1, #2

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Scaling present

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 d

Remarks on result:

other: Scaling present

Irritant / corrosive response data:

CLINICAL SKIN OBSERVATIONS:
1 h after removal of the dressing a well-defined erythema was observed in all animals which progressed into moderate to severe and persisted as very slight up to 10 or 14 d after treatment. A very slight to moderate oedema was recorded in all animals from the 1 h up to the 72 h reading or up to 10 d after treatment. Scaling was present in all animals 7 to 14 d after removal of the application patch. No alterations and no corrosive effects were observed on the treated skin.

REVERSIBILITY
With the exception of scaling in all three animals and a very slight erythema in the male, these effects were reversible and were no longer evident 14 days after treatment.

Other effects:

TOXIC EFFECTS OTHER THAN SKIN IRRITATION
- No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
- Body weights: the body weights of all rabbits were considered to be within the normal range of variability.
- No necropsy was performed on the animals sacrificed at termination of observation.

Irritant/corrosive response data for each animal at each observation time

Score at time point	Erythema	Edema
	Max. score: 4	Max. score: 4
1 h	2/2/2	3/2/2
24 h	3/3/3	3/3/2
48 h	3/3/3	2/2/2
72 h	3/3/3	2/2/2
7 d	1/1/1	1/0/1 *
10 d	1/1/1	1/0/1 *
14 d	1/0/0	0/0/0 *

* = scaling present

Interpretation of results:

other: Category 2 (irritant) based on CLP criteria

Conclusions:

Under the study conditions, the test substance is considered as irritant to rabbit skin.

Executive summary:

An in vivo study was conducted to determine the skin irritation potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS', according to OECD Guideline 404, in compliance with GLP. Three young adult New Zealand White rabbits (1 male and 2 females) were dermally exposed to 0.5 mL read across substance moistened with 0.5 mL purified water for 4 h under semi-occlusive conditions, followed by observation for 14 d. The skin reactions were scored according to the guideline. 1 h after removal of the dressing, well-defined erythema was observed in all animals which progressed into moderate to severe reaction and persisted as very slight up to 10 or 14 d after treatment. A very slight to moderate oedema was recorded in all animals after 1 h and up to 72 h reading as well up to 10 d following treatment. Scaling was present in all animals 7 to 14 d after removal of the application patch. No alterations and no corrosive effects were observed on the treated skin. Under the study conditions, the read across substance was considered to be irritating to rabbit skin (Arcelin, 2010).Based on the results of the read across study, a similar irritation response is expected for the test substance, 'di-C16 and C18-unsatd. AAEMIM-MS'.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Justification for type of information:

As per Annex XI of the REACH Regulation, the skin irritation has been assessed based on an in vivo skin irritation study conducted with the read across substance. In addition, the read across in vivo skin irritation study was conducted before the in vitro testing amendment came into force under the REACH Regulation in June 2016. ECHA Reference: https://echa.europa.eu/fr/-/reach-annexes-amended-registrants-to-use-alternative-test-methods.

Reason / purpose for cross-reference:

data waiving: supporting information

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From April 20, 2017 to May 12, 2017

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 (incl. QA statement)

Species:

other: EpiOcularTM tissue model (OCL-200-MatTek Corporation)

Strain:

other: Keratinocyte 4F1188

Details on test animals or tissues and environmental conditions:

Test system:
The EpiOcularTM tissue model (OCL-200-MatTek Corporation) is composed of stratified human keratinocytes in a three-dimensional structure, reflecting the morphology and function of the human corneal epithelium found in vivo.

MatTek’s EpiOcularTM system consists of normal, human-derived keratinocytes which have been cultured to form a stratified, squamous epithelium similar to that found in the cornea. Cultured on specially prepared cell culture inserts using serum-free culture medium, the cells differentiate to form a multi-layered structure with progressively stratified, but not cornified cells which closely parallel the corneal epithelium (for information see www.mattek.com). QC results for the specific lot of models received (Lot# 23779) were checked in-house for MatTek acceptance ranges with the following outcome:

- Morphology - PASS
- Tissue viability - PASS
- Skin barrier function (ET50 value for 0.3 % Triton X-100) where ET50 is the time taken for 0.3 % Triton X-100 to reduce the viability of the skin model to 50 % relative to the negative control) - PASS
- Sterility testing showed no contamination during long term antibiotic and antimycotic free culture - PASS

Vehicle:

other: PBS (Sterile Dulbecco’s Phosphate Buffered Saline)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20 µl of PBS (Sterile Dulbecco’s Phosphate Buffered Saline) plus 50 mg of test substance.

Duration of treatment / exposure:

6h ± 15 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation.

Duration of post- treatment incubation (in vitro):

25 ± 2 minutes

Number of animals or in vitro replicates:

Three tissues per condition (n=3).

Details on study design:

Preliminary test:
The test substance was first checked for its potential for MTT interference and solvent interference.

Main test overview:
Day 0: On the day of receipt, EpiOcularTM tissues were pre-incubated overnight at 37 °C, 5 % CO2, 95 % relative humidity.
Day 1: Exposure to and removal of test and reference substances (50 mg of test substance or 50 µl of reference substances for 6h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 hours ± 15 minutes post-treatment incubation). 
Day 2: End of MTT viability test, readings at 570 nm without reference filter.

Irritation parameter:

other: % viability

Run / experiment:

6 h exposure

Value:

15.6

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All validity criteria for the test were met:
- Criteria: the mean OD570 of the negative control (treated with sterile water) tissues is ≥ 0.8 and ≤ 2.5.
Result for the test: 1.334
- The mean of the positive control relative percentage viability is below 50% of negative control viability after 6 hours exposure.
Result for the test: 47.2
- The SD between three tissues replicates should not exceed 18 % in the same run (for negative and positive control tissues and tissues of test substances).
Results for the test:
NC: 3.88
PC: 4.976
Test substance: 4.582

Interpretation of results:

other: Inconclusive for the classification

Conclusions:

Under the study conditions, the percentage viability obtained was 15.6% and therefore, the test substance was classified as irritant to the human eye. However, based on the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage).

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16 and C18-unsatd. AAEMIM-MS', using Reconstructed human Cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. After pre-wetting the tissues with 20µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, test system was exposed to 50 mg of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation. After post-treatment incubation period, MTT test was performed with readings at 570 nm without reference filter, and percentage viability value for EpiOcularTM models exposed to the test substance relative to the negative control was calculated. The percentage viability obtained for negative control, positive control and test substance were calculated be 100%, 47.2% and 15.6% respectively. The test was considered to have met all the validity criteria. Since the percentage viability of the test substance was much below the threshold (>60%) indicating no irritation potential, the test substance was classified as irritant to the human eye by the study authors (XCellR8, 2017). However, based on the viability percentage in the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From August 31, 2017 to August 31, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

updated 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

Triplicate

Details on study design:

Preparation of Corneas: All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading: The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test substance and three corneas to the positive control substances.

Treatment of Corneas: The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test substance or control substance were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes. At the end of the exposure period the test substance and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein: Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations: After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology: The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Irritation parameter:

in vitro irritation score

Run / experiment:

10 minutes

Value:

4.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Inconclusive

Corneal epithelium condition: The corneas treated with the test substance were clear post treatment and partly cloudy post incubation. The corneas treated with the negative control substance were clear post treatment and post incubation. The corneas treated with the positive control substance were cloudy post treatment and post incubation.

Individual and mean corneal opacity and permeability measurements:

Treatment	Cornea Number	Opacity					Permeability (OD)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre-Treatment	Corrected Value		Corrected Value
Negative Control	1	3	3	4	1		0.007
	2	3	2	2	0		0.011
	3	4	2	2	0		0.007
					0.3*		0.008♦		0.5
Positive Control	4	6	40	37	31	30.7	0.794	0.786
	5	3	31	33	30	29.7	1.96	1.952
	6	2	24	26	24	23.7	1.885	1.877
						28•		1.538•	51.1
Test substance	10	3	6	6	3	2.7	0.099	0.091
	11	3	4	9	6	5.7	0.073	0.065
	12	1	3	4	3	2.7	0.014	0.006
						3.7•		0.054•	4.5

OD = Optical density * = Mean of the post-incubation − pre-treatment values ♦ = Mean permeability • = Mean corrected value

Corneal epithelium condition post treatment and post incubation

Treatment	Cornea number	Observation
		Post treatment	Post incubation
Negative Control	1	Clear	Clear
	2	Clear	Clear
	3	Clear	Clear
Positive Control	4	Cloudy	Cloudy
	5	Cloudy	Cloudy
	6	Cloudy	Cloudy
Test substance	10	Clear	Partly Cloudy
	11	Clear	Partly Cloudy
	12	Clear	Partly Cloudy

Results:

Treatment	In Vitro Irritancy Score
Test substance	4.5
Negative control	0.5
Positive control	51.1

Interpretation of results:

other: Inconclusive for classification

Conclusions:

Under the study conditions, eye irritation potential of the test substance was determined to be inconclusive based on bovine corneal opacity and permeability test (IVIS score – 4.5).

Executive summary:

An in vitro study was conducted to determine the eye irritation potential of the read across substance, ‘di-C16-18-satd. and C18-24 -unsatd. AAEMIM-MS' (active: 100%), using Bovine Corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (750 µL), negative control (Sodium chloride 0.9% w/v) and positive control (Ethanol) substances at 32 ± 1ºC for 120 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In vitro Irritancy Score (IVIS). The positive control IVIS was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of <3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The read across substance IVIS score obtained was 4.5, which is well below the corrosive limit of 55 and slightly above the non-corrosive limit of 3; therefore no predictions could be made. Under the study conditions, no prediction of eye irritation could be made for the read across substance (Envigo, 2018). Based on the results of the read across study, similar inconclusive results can be expected for the test substance, 'di-C16 and C18-unsatd. AAEMIM-MS'.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin

An in vivo study was conducted to determine the skin irritation potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS', according to OECD Guideline 404, in compliance with GLP. Three young adult New Zealand White rabbits (1 male and 2 females) were dermally exposed to 0.5 mL read across substance moistened with 0.5 mL purified water for 4 h under semi-occlusive conditions, followed by observation for 14 d. The skin reactions were scored according to the guideline. 1 h after removal of the dressing, well-defined erythema was observed in all animals which progressed into moderate to severe reaction and persisted as very slight up to 10 or 14 d after treatment. A very slight to moderate oedema was recorded in all animals after 1 h and up to 72 h reading as well up to 10 d following treatment. Scaling was present in all animals 7 to 14 d after removal of the application patch. No alterations and no corrosive effects were observed on the treated skin. Under the study conditions, the read across substance was considered to be irritating to rabbit skin (Arcelin, 2010). Based on the results of the read across study, a similar irritation response is expected for the test substance, 'di-C16 and C18-unsatd. AAEMIM-MS'.

 

Eye:

Study 1:

An in vitro study was conducted to determine the eye irritation potential of the test substance, ‘di-C16 and C18-unsatd. AAEMIM-MS', using Reconstructed human Cornea-like Epithelium (RhCE), according to OECD Guideline 492, in compliance with GLP. EpiOcularTM tissues were pre-incubated overnight at 37°C, 5% CO2, ≥95% RH. After pre-wetting the tissues with 20µL PBS (Sterile Dulbecco’s Phosphate Buffered Saline) for 30 ± 2 min, test system was exposed to 50 mg of the test substance or reference substances (negative control: sterile water; positive control: methyl acetate) for 6 h ± 15 minutes, followed by a 25 ± 2 minutes post-treatment immersion, and 18 h ± 15 minutes post-treatment incubation. After post-treatment incubation period, MTT test was performed with readings at 570 nm without reference filter, and percentage viability value for EpiOcularTM models exposed to the test substance relative to the negative control was calculated. The percentage viability obtained for negative control, positive control and test substance were calculated be 100%, 47.2% and 15.6% respectively. The test was considered to have met all the validity criteria. Since the percentage viability of the test substance was much below the threshold (>60%) indicating no irritation potential, the test substance was classified as irritant to the human eye by the study authors (XCellR8, 2017). However, based on the viability percentage in the current assay it is not possible to differentiate between GHS class 1 and GHS class 2 (degree of stromal damage), hence, the classification for the test substance is considered to be inconclusive.

Study 2:

An in vitro study was conducted to determine the eye irritation potential of the read across substance, ‘di-C16-18-satd. and C18-24 -unsatd. AAEMIM-MS' (active: 100%), using Bovine Corneal Opacity Test (BCOP), according to OECD Guideline 437 and EU Method B.47, in compliance with GLP. Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. Preparation, selection and opacity reading of the corneas were performed as per guideline. Prepared corneas in triplicates were treated with each, test substance (750 µL), negative control (Sodium chloride 0.9% w/v) and positive control (Ethanol) substances at 32 ± 1ºC for 120 minutes. At the end of the exposure period the test substance and control substances were removed from the anterior chamber and the cornea was rinsed three times with fresh complete Eagle’s Minimum Essential Medium (EMEM) containing phenol red before a final rinse with complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1ºC for 90 minutes. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In vitro Irritancy Score (IVIS). The positive control IVIS was within the range of 31.6 to 58.7. The positive control acceptance criterion was therefore satisfied. The negative control resulted in opacity of <3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied. The read across substance IVIS score obtained was 4.5, which is well below the corrosive limit of 55 and slightly above the non-corrosive limit of 3; therefore no predictions could be made. Under the study conditions, no prediction of eye irritation could be made for the read across substance (Envigo, 2018). Based on the results of the read across study, similar inconclusive results can be expected for the test substance, ' di-C16 and C18-unsatd. AAEMIM-MS' in a BCOP assay; hence further testing was conducted.

Based on the available in vitro eye irritation studies were conducted with test substance and read across substance, no clear conclusions could be drawn as per the Guidelines. However, given the IVIS score from BCOP study with read across substance, which was closer to the non-corrosive limit and considering that the percentage viability from the RhCE with test substance is low, indicates that the test substance, 'di-C16 and C18 -unsatd. AAEMIM-MS' can be considered to be more likely to be irritating to the eyes (in a worst case).

Justification for classification or non-classification

Skin:

Based on the results of the read across in vivo skin irritation study, the test substance, 'di-C16 and C18-unsatd. AAEMIM-MS' warrants ’Skin irritant Category 2; H315: Causes skin irritation’ classification according to EU CLP criteria (Regulation EC 1272/2008).

Eye:

Based on the results of read across and test substance based in vitro assays and as a conservative approach, the test substance, 'di-C16 and C18 -unsatd. AAEMIM-MS', is considered to warrant 'Eye Irrit. 2: H319- causes serious eye irritation’ classification according to the EU CLP criteria (Regulation 1272/2008/EC).