Reaction mass of Tetrasodium 3-{[5-({4-[alkyl(3-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)amino]-6-fluoro- heteromonocycle-2-yl}amino)-2-sulfonatophenyl]diazenyl}-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate and Trisodium 3-[{5-[(4-{[3-(ethenylsulfonyl)phenyl](alkyl)amino}-6-fluoro-heteromonocycle-2-yl)amino]-2-sulfonatophenyl}diazenyl]-4-hydroxy-5-(alkanoylamino)naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

Acute dermal irritation in rabbit (OECD 404): Negative (not irritatting)
In vitro eye irritation (BCOP, OECD 437): No prediction of eye irritation can be made.
In vivo eye irritation (OECD 405): Negative (not irritating)

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 04 August 2015 and 07 August 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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 male New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.59 or 2.68 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 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 Envigo RMS (UK) Limited, 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 12 hours continuous light and 12 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, no further testing was needed.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Remarks:

test item moistened with 0.5 ml of distilled water.

Controls:

no

Amount / concentration applied:

0.5 g

Duration of treatment / exposure:

Four hours

Observation period:

72 hours

Number of animals:

Two males

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.

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.

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

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.
 
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 theGlobally Harmonized Systemof Classification and Labelling of Chemicals.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: Not applicable

Irritant / corrosive response data:

Red colored staining, caused by the test item but not preventing evaluation of skin responses, was noted at both treated skin sites at all observations.
No evidence of skin irritation was noted during the study.

Other effects:

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

Individual Skin Reactions

Skin Reaction	Observation Time (following patch removal)	Individual Scores		Total
		Rabbit Number and Sex
		75165Male	75169Male
Erythema/Eschar Formation	Immediately	0STA	0STA	(0 )
	1 Hour	0STA	0STA	( 0 )
	24 Hours	0STA	0STA	0
	48 Hours	0STA	0STA	( 0 )
	72 Hours	0STA	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) :        0
Primary Irritation Index (S/4)              :        0/4 = 0.0
Classification                                       :        NON‑IRRITANT

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

STA =    Red colored staining

Individual Body Weights and Body Weight Change

Rabbit Number and Sex	Individual Body Weight (kg)		Body Weight Change (kg)
	Day 0	Day 3
75165 Male	2.68	2.71	0.03
75169 Male	2.59	2.61	0.02

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 0.0 and was classified as non 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 the Globally Harmonized System of Classification and Labelling of Chemicals.

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 male rabbits produced no evidence of skin irritation.

 

Conclusion

The test item produced a primary irritation index of0.0and was classified as non‑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 the Globally Harmonized Systemof Classification and Labelling of Chemicals.

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:

The study was conducted between 10 November 2015 and 19 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MAFF, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Animal Information
Two male New Zealand White (Hsdlf:NZW) strain rabbits were supplied by Envigo RMS (UK) Limited, Leicestershire, UK. At the start of the study the animals weighed 2.93 or 3.08 kg and were 12 to 20 weeks old. After an acclimatization period of at least 5 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.

Animal Care and Husbandry
The animals were individually housed in suspended cages. Free access to mains drinking water and food (2930C Teklad Global Rabbit diet supplied by Envigo RMS (UK) Limited, 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 12 hours continuous light and 12 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.

Vehicle:

unchanged (no vehicle)

Remarks:

Test item was used as supplied.

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 65 mg (as measured by gently compacting the required volume into an adapted syringe).

Duration of treatment / exposure:

The test item was not removed.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

Two males

Details on study design:

Experimental Design and Study Conduct
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 65 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 according to a six point scale.

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.

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

Irritation parameter:

cornea opacity score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

cornea opacity score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

4

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

iris score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24,48 and 72 hours

Score:

0

Max. score:

2

Reversibility:

other: No effects observed

Irritation parameter:

conjunctivae score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

conjunctivae score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Remarks:

75272 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

chemosis score

Remarks:

75263 Male

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0.666

Max. score:

4

Reversibility:

fully reversible within: 72 hours

Irritant / corrosive response data:

Ocular Reactions
There was no initial pain reaction, in either animal, following instillation of the test item.
Red colored staining of the fur around the treated eye was noted in both animals at all observations.
No corneal or iridial effects were noted during the study.
Moderate conjunctival irritation was noted in both treated eyes 1 hour after treatment. Moderate conjunctival irritation was noted in one treated eye with minimal conjunctival irritation noted in the other treated eye at the 24 Hour observation. Minimal conjunctival irritation was noted in both treated eyes at the 48 Hour observation.
Both treated eyes appeared normal at the 72 Hour observation.

Other effects:

Body Weight
One animal showed no gain in body weight and the other animal showed expected gain in body weight during the study.

Individual Scores and Individual Total Scores for Ocular Irritation

Rabbit Number and Sex	75272Male				75263Male
	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	1	1	0	2	2	1	0
B = Chemosis	1	1	1	0	2	1	1	0
C = Discharge	1Sf	1Sf	1Sf	0Sf	1Sf	1Sf	0Sf	0Sf
Score (A + B + C) x 2	8	6	6	0	10	8	4	0
Total Score	8	6	6	0	10	8	4	0

IPR=Initial pain reaction

Sf = Red colored staining of fur around treated eye

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
75272Male	8	6	6	0
75263Male	10	8	4	0
Group Total	18	14	10	0
Group Mean Score	9.0	7.0	5.0	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
75272Male	2.93	2.99	0.06
75263Male	3.08	3.08	0.00

Interpretation of results:

not classified

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 the Globally Harmonized System of Classification and Labelling of Chemicals.

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 male 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 amildirritant (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 the Globally Harmonized Systemof Classification and Labelling of Chemicals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Firstly , only the in vivo study (OECD 404) was performed as an acute dermal toxicity study (OECD 402) was already performed. The results (OECD 402) does not indicates skin irritation up to the limit dose level (2000 mg/kg body weight). This is the reason why we directly did the in vivo study instead of perform the in vitro test first. The acute dermal irritation in rabbits, (OECD 404), test is negative therefore the test item is not irritating in rabbit skin.

Concerning the eye irritation, one in vitro (OECD 437) and one in vivo (OECD 405) test were performed.

In the BCOP experiment (OECD 437) performed with FAT 40870/A TE, the conclusion was: No prediction of eye irritation can be made.

Then an in vivo experiment (OECD 405) was performed to assess the irritancy potential of the test item to the eye of the New Zealand White rabbit. 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 Calandraclassification system.

The test item does not meet the criteria for classification according to theGlobally Harmonized Systemof Classification and Labelling of Chemicals.

In conclusion, the test item is not irritant to eyes and skin.

Justification for selection of skin irritation / corrosion endpoint:
Only this study is available and it is Klimisch 1.
The in vivo acute dermal irritation in rabbits was performed (OECD 404) and was found to be negative (not irritating)
As an acute dermal toxicity study (OECD 402) was performed, the in vitro skin irriation/corrosion does not need to be performed as the acute dermal study does not indicate skin irritation up to the limit dose level (2000 mg/kg body weight).

Justification for selection of eye irritation endpoint:
No prediction of eye irritation can be made according to the results of the in vitro study on eyes (BCOP, OECD 437)
The in vivo test (OECD 405, acute eye irritaion in the rabbit) indicate that the test item in not irritant.
The in vivo study is selected as Key study and the study is Klimisch 1.

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).