Reactions mass of N-{2-[(4-bromo-6-substituted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-methoxyalkyl)amino]phenyl}acetamide) and N-{2-[(4-bromo-6-substiuted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-hydroxy-3-phenoxypropyl)amino]phenyl}acetamide

Endpoint summary

Description of key information

In vitro skin corrosion: Negative
In vitro skin irritation: Negative
In vitro eye irritation (BCOP): Negative.
In vivo eye irritation: Negative

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:

10 June 2014 to 13 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: 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:

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 2.90 and 2.95 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:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.5 g of the test item, moistened sufficiently with 0.5 mL of distilled water

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

2

Details on study design:

On the day before the test two rabbits were 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. At each test site a quantity of 0.5 g of the test item, moistened sufficiently with 0.5 mL of distilled water was introduced 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.

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal: 74369 Female

Time point:

other: Highest score at 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Remarks on result:

other: Black staining observed up to 1 hour after patch removal

Irritation parameter:

other: Erythema/Eschar Formation

Basis:

animal: 74370 Female

Time point:

other: Gighest score at 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No evidence of skin irritation

Remarks on result:

other: Black staining observed up to 72 hours after patch removal

Irritation parameter:

other: Oedema Formation

Basis:

animal: 74369 Female

Time point:

other: Highest score at 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No evidence of skin irritation

Irritation parameter:

other: Oedema Formation

Basis:

animal: 74370 Female

Time point:

other: Highest score at 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No evidence of skin irritation

Irritant / corrosive response data:

The individual scores for erythema/eschar and edema are given in Table 1.

Black staining, not preventing evaluation of skin responses, was noted at both treated skin sites during the study.

Very slight erythema was noted at one treated skin site at the 24 Hour observation.

No evidence of skin irritation was noted during the study.

Other effects:

Individual body weights and body weight change are given in Table 2.

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

Interpretation of Results
Calculation of Primary Irritation Index and Grading of Irritancy Potential Using the Draize Scheme

 

The scores for erythema and edema at the 24 and 72‑Hour readings were totaled for the two test rabbits (8 values) and this total was divided by 4 to give the primary irritation index of the test item. The test item was classified according to the following scheme devised byDraize, J.H. (1959):

 

Primary Irritation Index	Classification of Irritancy
0	Non-irritant
> 0 to 2	Mild irritant
> 2 to 5	Moderate irritant
> 5 to 8	Severe irritant

 

If irreversible alteration of the dermal tissue is noted in any rabbit, as judged by the Study Director, which include ulceration and clear necrosis or signs of scar tissue, the test item is classified as corrosive to rabbit skin. Classification according to Draize may, therefore, not be applicable.

 

The results were also interpreted according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Table 1     Individual Skin Reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores – Rabbit Number and Sex		Total
		74369 Female	74370 Female
Erythema / Eschar Formation	Immediately	0STA	0STA	(0 )
	1 Hour	0STA	0STA	( 0 )
	24 Hours	1	0STA	1
	48 Hours	0	0STA	( 0 )
	72 Hours	0	0STA	0
Edema Formation	Immediately	0	0	( 0 )
	1 Hour	0	0	( 0 )
	24 Hours	0	0	0
	48 Hours	0	0	( 0 )
	72 Hours	0	0	0
Sum of 24 and 72‑Hour Readings (S) :        1
Primary Irritation Index (S/4)              :        1/4 = 0.3
Classification                                       :        MILD IRRITANT

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

STA =   Black staining

Table 2     Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
74369 Female	2.95	3.05	0.10
74370 Female	2.90	2.98	0.08

Interpretation of results:

other: The test item produced a primary irritation index of 0.3 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme.

Remarks:

Criteria used for interpretation of results: other: Draize classification scheme.

Conclusions:

The test item produced a primary irritation index of 0.3 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

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 skin of the New Zealand White rabbit.

 

Results

A single 4‑Hour, semi‑occluded application of the test item to the intact skin of two rabbits produced very slight erythema at one treated skin site at the 24‑Hour observation. No evidence of skin irritation was noted at the other treated skin site during the study

 

Conclusion

The test item produced a primary irritation index of 0.3 and was classified as a mild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Testing was conducted between 16th June and 7th July 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animals and Animal Husbandry:
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 2.31 or 2.55 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.

Vehicle:

unchanged (no vehicle)

Controls:

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

Amount / concentration applied:

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
A volume of 0.1 mL of the test item, which was found to weigh approximately 50 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.

- Concentration (if solution):
Undiluted and used as supplied

VEHICLE
For the purpose of the study the test item was used as supplied

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

Approximately 1 hour and 24, 48 and 72 hours following treatment

Number of animals or in vitro replicates:

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.

Details on study design:

PROCEDURE:
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 50 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.

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) (please see the any other information on materials and methods section, including tables) to find the Draize scoring system.

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.

An additional observation was made on Day 7 to assess the reversibility of the ocular effects.

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

Irritation parameter:

cornea opacity score

Basis:

animal: 74403 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Basis:

animal: 74448 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

1.67

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Basis:

animal: 74403 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: no effects observed

Irritation parameter:

iris score

Basis:

animal: 74448 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

0.33

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 74403 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal: 74448 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 74403 Female

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritation parameter:

chemosis score

Basis:

animal: 74448 Male

Time point:

other: Mean 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Individual scores for ocular irritation are given in Table 1* (please see the any other information on results section, including tables)

Black staining of the fur was noted around one treated eye throughout the study.

No corneal effects were noted during the study.

Iridial inflammation was noted in both treated eyes 1 hour after treatment and in one treated eye at the 24 Hour observation.

Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment with minimal conjunctival irritation noted at the 24, 48 and 72 Hour observations.

Both treated eyes appeared normal at the 7 Day observation.

* Table 1 can be found in the any other information on results section (including tables)

Other effects:

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

Table 1     Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	74403Female					74448Male
	IPR= 0					IPR = 0
Time After Treatment	1 Hour	24 Hours	48 Hours	72 Hours	7 Days	1 Hour	24 Hours	48 Hours	72 Hours	7 Days
CORNEA
E = Degree of Opacity	0	0	0	0	0	0	0	0	0	0
F = Area of Cornea Involved	0	0	0	0	0	0	0	0	0	0
Score (E x F) x 5	0	0	0	0	0	0	0	0	0	0
IRIS
D	1	0	0	0	0	1	1	0	0	0
Score (D x 5)	5	0	0	0	0	5	5	0	0	0
CONJUNCTIVAE
A = Redness	1	1	1	1	0	2	2	1	1	0
B = Chemosis	1	1	1	1	0	1	1	1	1	0
C = Discharge	2Sf	1Sf	1Sf	0Sf	0Sf	1	0	0	0	0
Score (A + B + C) x 2	8	6	6	4	0	8	6	4	4	0
Total Score	13	6	6	4	0	13	11	4	4	0

 

IPR= Initial pain reaction

Sf = Black staining of the fur around the treated eye

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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 iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 7‑Day observation.

 

 Conclusion:

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION/CORROSION:

1) The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. .

 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre‑labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues. 

 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 562 nm.

 

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

 

Results:

The relative mean viability of the test item treated tissues was106.6% after the 15‑Minute exposure period and 42 hours post‑exposure incubation period.

 

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

 

Conclusion:

The test item was classified as non-irritant. The following classification criteria apply:

EU DSD & CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

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

 

Results

A single 4‑Hour, semi‑occluded application of the test item to the intact skin of two rabbits produced very slight erythema at one treated skin site at the 24‑Hour observation. No evidence of skin irritation was noted at the other treated skin site during the study

 

Conclusion

The test item produced a primary irritation index of0.3and was classified as amild irritant to rabbit skin according to the Draize classification scheme. No corrosive effects were noted.

 

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.

EYE IRRITATION:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and bioitem function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability.

  

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. 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 anIn VitroIrritancy Score (IVIS).

 

Results:

TheIn Vitroirritancy scores are summarized as follows:

 

Treatment	In VitroIrritancy Score
Test Item	9.3
Negative Control	1.5
Positive Control	93.0

 

Conclusion:

No prediction of eye irritation can be made.

 

The results of this study have identified the test item as not causing serious eye damage, but they do not permit conclusion that the test item does not require classification for eye irritation.

2)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 iridial inflammation and moderate conjunctival irritation. Both treated eyes appeared normal at the 7‑Day observation.

 

 Conclusion:

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.

Justification for selection of skin irritation / corrosion endpoint:
- The conclusion of the in vitro test (OECD 439) is negative (non-irritant).
- The conclusion of the in vibo test (OECD 404) is positive (slightly irritant) according to the Draize classification.
The test item produced a primary irritation index of 0.3 and was classified as a mild irritant to rabbit skin according to the Draize classification
scheme. No corrosive effects were noted.
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.

Justification for selection of eye irritation endpoint:
- The in vitro test (OECD 437) is negative.
- The in vivo test (OECD 405) is negative.

Justification for classification or non-classification

Based on the above mentioned results, the substance does not need to be classified according to CLP regulation (Regulation EC No.1272/2008) and DSD (Directive 67/548/EEC).