2-Butenoic acid, 1-ethyl-2-methylpropyl ester, (2E)-

Endpoint summary

Description of key information

The test substance, TM 12-209, was assessed for skin corrosion and irritation and for eye irritation according to internationally recognized guidelines both in in vitro and in vivo tests.  Results indicated that the test item was considered not to be corrosive or a skin irritant. The test substance did not meet the requirements of classification as an eye irritant. 

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 May 2014 and 03 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 404 using the in vivo Acute Dermal Irritation/Corrosion method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Harlan Laboratories UK Ltd., Leicestershire, UK. At the start of the study the animals weighed 2.92 to 3.10 kg and were twelve to twenty weeks old. After an acclimatization period of at least five days each animal was given a number unique within the study which was written with a black indelible marker pen on the inner surface of the ear and on the cage label.

The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet and drinking water were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 17 to 23 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.5 mL of the test item was applied directly to the skin.

Test Item Formulation
For the purpose of the study the test item was used as supplied. The absorption of the test item was not determined.

Duration of treatment / exposure:

Four hours

Observation period:

14 days

Number of animals:

Three

Details on study design:

On the day before the test each of a group of three rabbits was clipped free of fur from the dorsal/flank area using veterinary clippers. Only animals with a healthy intact epidermis by gross observation were selected for the study.

On the day of the test a suitable test site was selected on the back of each rabbit. A quantity of 0.5 mL of the undiluted test item was applied directly to the skin under a 2.5 cm x 2.5 cm cotton gauze patch. The patch was secured in position with a strip of surgical adhesive tape. To prevent the animals interfering with the patches, the trunk of each rabbit was wrapped in an elasticated corset and the animals were returned to their cages for the duration of the exposure period.

Four hours after application the corset and patches were removed from each animal and any residual test item removed by gentle swabbing with cotton wool soaked in distilled water.

Immediately following removal of the patches and approximately 1, 24, 48 and 72 hours later, the test sites were examined for evidence of primary irritation and scored.

Any other skin reactions and clinical signs of toxicity, if present, were also recorded.

Additional observations were made on Days 7 and 14 to assess the reversibility of skin reactions.

Individual body weights were recorded on Day 0 (the day of dosing) and at the end of the observation period.

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal #1

Remarks:

74312 Male

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Erythema extended approximately 10 mm around the treated skin site. At the 7 day timepoint a score of 1 was given with slight desquamation noted.

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal #2

Remarks:

74313 Male

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Erythema extended approximately 10 mm around the treated skin site. At the 7 day timepoint a score of 1 was given with slight desquamation noted.

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal #3

Remarks:

74314 Male

Time point:

other: 24, 48 and 72 hours

Score:

1.67

Max. score:

4

Reversibility:

fully reversible within: 14 days

Remarks on result:

other: Erythema extended approximately 10 mm around the treated skin site. At the 7 day timepoint a score of 1 was given with slight desquamation noted.

Irritation parameter:

edema score

Basis:

animal #1

Remarks:

74312 Male

Time point:

other: 24, 48 and 72 hours

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #2

Remarks:

74313 Male

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

edema score

Basis:

animal #3

Remarks:

74314 Male

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Well-defined erythema and very slight or slight edema were noted at all treated skin sites at the 24-Hour observation. Well defined erythema and very slight or slight edema were noted at two treated skin sites with very slight erythema and very slight edema noted at one treated skin site at the 48 Hour observation. Well defined erythema and very slight or slight edema were noted at all treated skin sites at the 72 Hour observation. The erythema extended approximately 10 mm around all treated skin sites at the 24 and 48 Hour observations. Very slight erythema and slight desquamation were noted at all treated skin sites at the 7 Day observation. All treated skin sites appeared normal at the 14 Day observation.

Other effects:

Body Weight
All animals showed expected gain in body weight during the study.

Individual Skin Reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores – Rabbit Number and Sex			Total
		74312 Male	74313 Male	74314 Male
Erythema/Eschar Formation	Immediately	0	0	0	(0 )
	1 Hour	0	0	0	( 0 )
	24 Hours	2R	2R	2R	6
	48 Hours	2R	2R	1R	( 5 )
	72 Hours	2	2	2	6
	7 Days	1D	1D	1D	( 3 )
	14 Days	0	0	0	( 0 )
Edema Formation	Immediately	0	0	0	( 0 )
	1 Hour	0	0	0	( 0 )
	24 Hours	2	1	1	4
	48 Hours	2	1	1	( 4 )
	72 Hours	2	1	1	4
	7 Days	0	0	0	( 0 )
	14 Days	0	0	0	( 0 )
Sum of 24 and 72-Hour Readings (S)           :          20
Primary Irritation Index (S/6)            :          20/6 = 3.3
Classification                                     :          MODERATE IRRITANT

(   ) = Total values not used for calculation of primary irritation index

R = Erythema extended approximately 10 mm around the treated skin site

D = Slight desquamation

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 14
74312 Male	3.04	3.38	0.34
74313 Male	3.10	3.47	0.37
74314 Male	2.92	3.40	0.48

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a primary irritation index of 3.3 and was classified as a moderate irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

The test item meets the criteria for classification as a Mild irritant (Category 3) according to the Globally Harmonized System of Classification and Labelling of Chemicals.

The test item does not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

The dermal irritation potential of the test substance, TM 12-209, was assessed as not irritating according to OECD Test Guideline 404 using the in vivo Acute Dermal Irritation/Corrosion method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted on 05 June 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Eyes from adult cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin and Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

0.75 mL of test item was applied.

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

120 minutes

Number of animals or in vitro replicates:

Not applicable

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 (epithelial side uppermost) 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 (MEM) 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 Raeding
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items 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 item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. 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 and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
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 (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 medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 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:

overall irritation score

Basis:

mean

Time point:

other: 120

Score:

7.1

Reversibility:

not specified

Irritant / corrosive response data:

Corneal Epithelium Condition
The condition of each cornea post treatment and at the final opacity measurement are provided. The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

In virto Irritancy Score
The In Vitro irritancy scores are summarized as follows:
Test Item: 7.1
Negative Control: 0.6
Positive Control: 40.3

Criterion for an acceptable test

The positive control In Vitro Irritancy Score was within the range of 22.4 to 60.3. The positive control acceptance criterion was therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

	Cornea No.	Opacity				Permeability (OD)		In vitro irritancy score
		Pre-treatment	Post-treatment	Post-treatment – pre-treatment	Corrected value		Corrected value
Negative control	2	2	3	1		0.032
	4	2	2	0		0.024
	8	2	2	0		0.001
				0.3*		0.019**		0.6
Positive control	1	3	23	20	19.7	1.343	1.324
	3	3	25	22	21.7	1.201	1.182
	7	3	26	23	22.7	1.313	1.294
					21.3***		1.267***	40.3
Test item	5	1	9	8	707	0.101	0.082
	6	3	12	9	8.7	0.068	0.049
	9	2	5	3	2.7	0.041	0.022
					6.3***		0.051***	7.1

OD = Optical density

* = Mean of the post-treatment-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	2	Clear	Clear
	4	Clear	Clear
	8	Clear	Clear
Positive Control	1	Cloudy	Cloudy
	3	Cloudy	Cloudy
	7	Cloudy	cloudy
Test Item	5	Clear	Clear
	6	Clear	Clear
	9	clear	Clear

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant. The result of this study has identified the test item as not causing serious eye damage, but this does not permit conclusion that the test item does not require classification for eye irritation.

Executive summary:

The eye irritancy potential of the test substance, TM 12-209, was assessed as not irritating according to OECD Test Guideline 437 using the Bovine Corneal Opacity and Permeability Assay method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Corrosion/Irritation

Skin corrosion is defined as the production of irreversible damage to the skin, namely visible necrosis through the epidermis and into the dermis following the application of a test substance for up to 4 hours.

Corrosive reactions are typified by ulcers, bleeding, bloody scabs and, by the end of observations at 14 days, by discolouration due to blanching of the skin, complete areas of alopecia and scars.

Skin irritation is defined as the production of reversible damage to the skin following the application of a test substance for up to 4 hours.

Several factors are considered in determining the corrosion and irritation potential of substances before in vivo testing is undertaken. The potential for chemical induced skin corrosion is an important consideration in establishing procedures for the safe handling, packing and transport of chemicals. 

 

To this end, an in vitro study was performed to assess the potential for skin corrosion using the EPISKIN^TM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes.

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.

This study was designed to be compatible with OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test” (adopted 13 April 2004).

The test item is applied topically to the stratum corneum surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The test is based on the experience that corrosive chemicals are cytotoxic after a short term exposure to the EPISKIN^TM model. Corrosive chemicals are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic conversion of the vital dye MTT to formazan by viable cells in the test item treated cultures relative to the negative control.

Results showed that the test item did not reduce MTT and relative mean viabilities of the test item treated tissues were 115.1 % after 3 minutes exposure and 132.3 % after 240 minutes exposure. The test item should not therefore be classified as corrosive.

 

The test item was also assessed for skin irritation using the EPISKIN^TM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. As described above, the principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-Hour post-exposure incubation period may also be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point can be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Following a full validation study, the EpiSkin^TM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

The test item was applied topically as the dermal route is the most likely exposure route and the results of the study are believed to be of value in predicting the likely skin irritancy potential to man.

This study was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 439 “In Vitro Skin Irritation” (adopted 22 July 2010).

The relative mean viability of the test item treated tissues was 82.2 % after a 15-Minute exposure period and 42 hours post-exposure incubation period. From these results the test item was classified as non-irritant.

 

Further to this, an in vivo study was performed to assess the irritancy potential of the test item following a single, 4-hour, semi-occluded application to the intact rabbit skin. This study was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 404 “Acute Dermal Irritation/Corrosion” (adopted 24 April 2002).

Well-defined erythema and very slight or slight edema were noted at all treated skin sites at the 24-Hour observation. Well defined erythema and very slight or slight edema were noted at two treated skin sites with very slight erythema and very slight edema noted at one treated skin site at the 48 Hour observation. Well defined erythema and very slight or slight edema were noted at all treated skin sites at the 72 Hour observation. The erythema extended approximately 10 mm around all treated skin sites at the 24 and 48 Hour observations. Very slight erythema and slight desquamation were noted at all treated skin sites at the 7 Day observation. The mean value was <2.3 for erythema (in all animals) and for edema (in one animal) at 24, 48 and 72 hrs.

However, all treated skin sites appeared normal at the 14-Day observation, therefore according to OECD Test Guideline 404; the dermal irritation potential of test material was assessed as not irritating.

 

 

Eye Irritation

Serious eye damage is defined as the production of tissue damage in the eye, or serious physical decay of vision following application of a test substance to the anterior surface of the eye, which is not fully reversible within 21 days of application.

Eye irritation means the production of changes in the eye following application of test substance to the anterior surface of the eye, which are fully reversible within 21 days of application.

The classification system for substances involves a tired testing and evaluation scheme and a number of factors are considered in determining eye irritation or serious eye damage.

 

Initially, a study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. Two endpoints, namely 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). Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

This study was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 437 (2009) “Bovine Corneal Opacity and Permeability Assay”.

The results showed that the corneas treated with the test item were clear post incubation and therefore the test item was considered not to be an ocular corrosive or severe irritant.

 

Further to this, a study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. This study was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 02 October 2012).

 

The results showed that a single application of the test item to the non-irrigated eye of three rabbits produced iridial inflammation and moderate conjunctival irritation. Two treated eyes appeared normal at the 72‑Hour observation and one treated eye appeared normal at the 7‑Day observation. The test item does not meet the criteria for classification as an ocular corrosive or severe irritant.

Justification for selection of skin irritation / corrosion endpoint:
The study was conducted in vivo in an appropriate test species according to internationally recognised guidelines.

Justification for selection of eye irritation endpoint:
The study was conducted in vitro in an appropriate test system according to internationally recognised guidelines.

Justification for classification or non-classification

Skin Corrosion/Irritation

Substances are classified as being corrosive and irritant to skin according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. In vitro skin irritation and corrosion values are expressed as percentage relative mean viabilities, whilst in vivo irritation values are expressed as the mean value of erythema/eschar or edema in at least 2 of 3 tested animals.

Classification of in vitro irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-Hour post-exposure incubation period. If the relative mean tissue viability is ≤ 50 % the substance is predicted as being an irritant. Classification of in vitro corrosion potential is based on relative viabilities for each exposure time. At 3, 3/60, 60/240 and 240 minutes the relative mean tissue viability percentages are predicted as <35 (corrosive), ≥35/<35 (corrosive), ≥35/<35 (corrosive) and ≥35 respectively. Classification of in vivo irritation potential is that at least 2 of 3 animals tested having a mean score of ≥2.3 - ≤ 4.0 for erythema/eschar or edema formation.

Two in vitro tests were performed on the EPISKIN^TM Reconstructed Human Epidermis model for both irritation and corrosion potential according to internationally recognised guidelines and conducted according to GLP standards. Both of the in vitro tests did not meet the criteria for classification of test item, TM 12-209, and therefore is considered not to be corrosive or a skin irritant.

A further study performed in vivo was conducted according to internationally recognised guidelines and to GLP standards. This demonstrated well-defined erythema and very slight edema at all treated skin sites at the 24, 48 and 72 hour observations (with mean value <2.3 for erythema (in all animals) and for edema (in one animal)), however all treated skin sites appeared normal and had reversed at the 14-day observation. The test substance did not meet the criteria for classification according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures according to this study and is therefore not classified as a skin irritant.

Eye Irritation

Substances can be allocated to one of two categories based on irreversible effects on the eye (Category 1) and irritating to the eye (Category 2) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures. In vitro eye irritation values were expressed as the In Vitro Irritancy Score (IVIS), whilst in vivo irritation values were expressed as the mean value of corneal opacity, iritis, and conjunctival redness / oedema (chemosis) in at least 2 of 3 tested animals.

An in vitro test was performed using a Bovine Corneal Opacity and Permeability Assay. In this assay, an IVIS ≤3 results in no classification as an eye irritant. An IVIS of > 3 ≤55 indicates that no prediction can be made. An IVIS of > 55 results in a classification as a category 1 eye irritant.

The in vitro study performed according to internationally recognized guidelines and conducted to GLP on test item TM 12-209 gave an IVIS of 7.1 and therefore the test substance is not considered an ocular corrosive or severe irritant.

An in vivo study was performed according to internationally recognised guidelines and conducted to GLP standards. In this assay, a corneal opacity of ≥ 1 and / or iritis ≥ 1 and / or conjunctival redness ≥ 2 and conjunctival oedema (chemosis) ≥ 2 results in classification as an eye irritant.

The in vivo study demonstrated that a single application of the test item to the non-irrigated eye of three rabbits produced no corneal effects. Iridial inflammation was noted in one treated eye 1 hour after treatment with a mean score of < 1. Minimal conjunctival irritation was noted in all treated eyes at the 24 and 48‑hour observations and persisted in one treated eye at the 72‑hour observation, with a mean score < 2. Two treated eyes appeared normal at the 72‑hour observation and one treated eye appeared normal at the 7‑day observation. The test substance is not considered an ocular corrosive or severe irritant.