Reaction mass of 2-[[[2-[(2-aminoethyl)amino]ethyl]amino]carbonyl]-benzoic acid and 2,2' -[iminobis(2,1-ethanediyliminocarbonyl)]bis-benzoic acid and 7,8,9,10,11,12,20,21,22,23,24,25-dodecahydrodibenzo[i,t] [1,4,7,12,15,18] hexaazacyclodocosine-5,13,18,26(6H,19H)-tetrone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 09 September 2014 and 18 September 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-({2-[(2-aminoethyl)amino]ethyl}carbamoyl)benzoic acid; 2-{[2-({2-[(2-carboxyphenyl)formamido]ethyl}amino)ethyl]carbamoyl}benzoic acid; 3,6,9,18,21,24-hexaazatricyclo[24.4.0.0¹¹,¹⁶]triaconta-1(30),11,13,15,26,28-hexaene-2,10,17,25-tetrone

EC Number:

942-764-6

IUPAC Name:

2-({2-[(2-aminoethyl)amino]ethyl}carbamoyl)benzoic acid; 2-{[2-({2-[(2-carboxyphenyl)formamido]ethyl}amino)ethyl]carbamoyl}benzoic acid; 3,6,9,18,21,24-hexaazatricyclo[24.4.0.0¹¹,¹⁶]triaconta-1(30),11,13,15,26,28-hexaene-2,10,17,25-tetrone

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: 1,3-Isobenzofurandione, reaction products with diethylenetriamine
Purity: >86%
Physical state / Appearance: yellow powder
Storage Conditions: room temperature in the dark

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Two 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 2.54 or 2.97 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.

Justification
The rabbit is the preferred species of choice as historically used for irritation studies and is specified in the appropriate test guidelines. The number of animals used was the minimum required to achieve the objectives of the study. Testing was conducted in two animals and the response in those animals was such that exposure of a third animal would not affect classification of the test item, therefore, no further testing was needed.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: The left eye remained untreated and was used for control purposes.

Amount / concentration applied:

A volume of 0.1 mL of the test item, which was found to weigh approximately 75 mg was used.

Duration of treatment / exposure:

The test substance was not removed from the test eye.

Observation period (in vivo):

Assessment of ocular damage/irritation was made approximately 1 hour and 24, 48 and 72 hours following treatment.

Number of animals or in vitro replicates:

Two

Details on study design:

Immediately before the start of the test, both eyes of the provisionally selected test rabbits were examined for evidence of ocular irritation or defect with the aid of a light source from a standard ophthalmoscope. Only animals free of ocular damage were used.

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

A volume of 0.1 mL of the test item, which was found to weigh approximately 75 mg (as measured by gently compacting the required volume into an adapted syringe) was placed into the conjunctival sac of the right eye, formed by gently pulling the lower lid away from the eyeball. The upper and lower eyelids were held together for about one second immediately after treatment, to prevent loss of the test item, and then released. The left eye remained untreated and was used for control purposes. Immediately after administration of the test item, an assessment of the initial pain reaction was made.
Eight hours after test item application, a subcutaneous injection of post dose analgesia, buprenorphine 0.01 mg/kg and meloxicam 0.5 mg/kg, was administered to provide a continued therapeutic level of systemic analgesia. The treated animal was checked for signs of pain and suffering approximately 12 hours later. No further analgesia was required.

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

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

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

Any clinical signs of toxicity, if present, were also recorded.

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

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

Male 74697

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

other: Not classified

Results 2

Irritation parameter:

cornea opacity score

Remarks:

Male 74710

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Remarks on result:

other: Not classified

Results 3

Irritation parameter:

iris score

Remarks:

Male 74697

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Remarks on result:

other: Not classified

Results 4

Irritation parameter:

iris score

Remarks:

Male 74710

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: Not applicable

Results 5

Irritation parameter:

conjunctivae score

Remarks:

Male 74697

Basis:

mean

Remarks:

Redness

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Not classified

Results 6

Irritation parameter:

conjunctivae score

Remarks:

Male 74710

Basis:

mean

Remarks:

Redness

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Not classified

Results 7

Irritation parameter:

chemosis score

Remarks:

Male 74697

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Not classified

Results 8

Irritation parameter:

chemosis score

Remarks:

Male 74710

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.66

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Not classified

Results 9

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 1 hour

Score:

9

Max. score:

110

Reversibility:

fully reversible within: 72 hours

Results 10

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 24 hours

Score:

8

Max. score:

110

Reversibility:

fully reversible within: 72 hours

Results 11

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 48 hours

Score:

4

Max. score:

110

Reversibility:

fully reversible within: 72 hours

Remarks on result:

other: Classed as a mild irritant according to a modified Kay and Calandra classification system; not classified according to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Results 12

Irritation parameter:

maximum mean total score (MMTS)

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

110

Reversibility:

other: Not applicable

Irritant / corrosive response data:

No corneal or iridial effects were noted during the study.

Moderate conjunctival irritation was noted in both treated eyes 1 and 24 hours after treatment with minimal conjunctival irritation noted at the 48 Hour observation.

Both treated eyes appeared normal at the 72 Hour observation.

Other effects:

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

Any other information on results incl. tables

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	74697Male				74710Male
	IPR= 0				IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	1 Hour	24 Hours	48 Hours	72 Hours
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0
IRIS
D	0	0	0	0	0	0	0	0
Score (D x 5)	0	0	0	0	0	0	0	0
CONJUNCTIVAE
A = Redness	2	2	1	0	2	2	1	0
B = Chemosis	2	1	1	0	1	1	1	0
C = Discharge	1	1	0	0	1	1	0	0
Score (A + B + C) x 2	10	8	4	0	8	8	4	0
Total Score	10	8	4	0	8	8	4	0

IPR=Initial pain reaction

Individual Total Scores and Group Mean Scores for Ocular Irritation

Rabbit Number and Sex		Individual Total Scores At:
		1 Hour		24 Hours		48 Hours		72 Hours
74697Male		10		8		4		0
74710Male		8		8		4		0
Group Total		18		16		8		0
Group Mean Score		9.0		8.0		4.0		0.0

 

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
74697Male	2.54	2.61	0.07
74710Male	2.97	3.04	0.07

 

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class 4 on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.
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:

Introduction

The study was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit.

 

Results

A single application of the test item to the non-irrigated eye of two rabbits produced moderate conjunctival irritation. Both treated eyes appeared normal at the 72‑Hour observation.

 

Conclusion

The test item produced a maximum group mean score of 9.0 and was classified as a mild irritant (Class4on a 1 to 8 scale) to the rabbit eye according to a modified Kay and Calandra classification system.

 

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.