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

The test item did not induce toxic effects in the rat following an oral or a dermal administration at a level of 2000 mg/kg or following . The lack of mortality demonstrates the LD50 is greater than 2000 mg/kg body weight for the acute oral toxicity and for acute dermal toxicity.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 May 2015 and 27 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the body weight of the initially dosed animal.

The animals were housed in groups of up to four in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °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 (06:00 to 18:00) 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
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

For the purpose of the study the test item was freshly prepared, as required, as a solution in distilled water.

The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Doses:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.
A single animal was treated. In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated.
A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

No. of animals per sex per dose:

A single animal was treated. In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated.
A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose. In the absence of toxicity at a dose level of 2000 mg/kg, an additional group of animals was treated. A total of five animals were therefore treated at a dose level of 2000 mg/kg in the study.

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each dose group to confirm the survival of the previously dosed animals.

Clinical observations were made 30 minutes, 1, 2, and 4 hours after dosing and then daily for 14 days. Morbidity and mortality checks were made twice daily.

Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.

At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

No data

Preliminary study:

No data

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period. Red colored staining of the feces was noted in all animals from Day 1 and up to 6 days after dosing.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

None specified

Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Female	0	0	0	0	0F	0F	0F	0F	0F	0F	0	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	2-3 Female	0	0	0	0	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

F =   Red colored staining of the feces

Individual Body Weights and Body Weight Changes

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
2000	1-0 Female	170	192	204	22	12
	2-0 Female	164	198	208	34	10
	2-1 Female	175	209	217	34	8
	2-2 Female	179	194	201	15	7
	2-3 Female	165	207	217	42	10

Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Female	Killed Day 14	No abnormalities detected
	2-0 Female	Killed Day 14	No abnormalities detected
	2-1 Female	Killed Day 14	No abnormalities detected
	2-2 Female	Killed Day 14	No abnormalities detected
	2-3 Female	Killed Day 14	No abnormalities detected

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System - Unclassified).

Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

 

Methods

Following a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were given a single oral dose of test item, as asolutionindistilled water, at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

 

Results

Mortality. There were no deaths.

 

Clinical Observations. No signs of systemic toxicity were noted. Red colored staining of the feces was noted in all animals from Day 1 and up to 6 days after dosing.

 

Body Weight. All animals showed expected gains in body weight.

 

Necropsy. No abnormalities were noted at necropsy.

 

Conclusion

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System-Unclassified).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Study Klimsich 1.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 14 May 2015 and 28 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five male and five female Wistar (RccHan™:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.
The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24 Hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °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 (06:00 to 18:00) 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
Rats are the preferred species of choice as historically used for safety evaluation studies and are specified in the appropriate test guidelines.

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

the test item was moistened with distilled water.

Details on dermal exposure:

On the day before treatment the back and flanks of each animal were clipped free of hair.

Using available information on the toxicity of the test item, a group of five male and five female rats was treated with the test item at a dose level of 2000 mg/kg.

The appropriate amount of test item, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage. The animals were caged individually for the 24 Hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.

After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test item. The animals were returned to group housing for the remainder of the study period.

The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.

After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored.
Any other skin reactions, if present were also recorded.

Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.

At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Duration of exposure:

24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

Five per sex per dose

Control animals:

no

Details on study design:

For the purpose of the study the test item was weighed out according to each animal’s individual body weight and moistened with distilled water prior to application.
The absorption of the test item was not determined.

Statistics:

No data

Preliminary study:

Not applicable

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period. Dermal reactions Pink colored staining was noted at the test sites of all animals during the study. The staining persisted at the test sites of males until Day 7 and of females up t

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

No data

Individual Clinical Observations and Mortality Data

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4 Male	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-3 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-4 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

Individual Dermal Reactions - Males

Dose Level mg/kg	Animal Number and Sex	Observation	Effects Noted After Initiation of Exposure (Days)
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0 Male	Erythema	?s	?s	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	Hd	Ss	STASs	STASs	STASs	STA	STA	0	0	0	0	0	0	0
	1-1 Male	Erythema	?s	?s	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	Hd	Ss	STA	STA	STA	STA	STA	0	0	0	0	0	0	0
	1-2 Male	Erythema	?s	?s	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	Hd	0	STA	STA	STA	STA	STA	0	0	0	0	0	0	0
	1-3 Male	Erythema	?s	?s	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	Hd	Ss	STA	STA	STA	STA	STA	0	0	0	0	0	0	0
	1-4 Male	Erythema	?s	?s	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	Hd	Ss	STASs	STASs	STASs	STA	STA	0	0	0	0	0	0	0

0= No reactions

STA = Pink colored staining

?s = Pink colored staining prevented accurate evaluation of erythema

Hd = Hemorrhage of dermal capillaries

Ss = Small superficial scattered scabs

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

Executive summary:

Introduction

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.    

Methods

A group of ten animals (five males and five females) was given a single, 24 hour, semi‑occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.    

Results

Mortality. There were no deaths.  

Clinical Observations. There were no signs of systemic toxicity.  

Dermal Irritation. Pink colored staining was noted at the test sites of all animals during the study and prevented accurate evaluation of erythema at the test sites of all animals 1 and 2 days after dosing. Signs of dermal irritation noted were hemorrhage of the dermal capillaries, small superficial scattered scabs, glossy skin and/or scab lifting to reveal glossy skin.

 

Body Weight. Three females showed body weight loss during the first week with expected gain in body weight during the second week. The remaining animals showed expected gains in body weight over the study period.  

Necropsy. No abnormalities were noted at necropsy.    

Conclusion

The acute dermal median lethal dose (LD₅₀) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Study Klimisch 1.

Additional information

Two differents studies were performed to assess the acute oral toxicity and the acute dermal toxicity of the test item in the Wistar strain rat.

For the acute oral test, following a sighting test at a dose level of 2000 mg/kg, an additional four fasted female animals were given a single oral dose of test item, as a solution in distilled water, at a dose level of 2000 mg/kg body weight. 

- No mortality occured during the test, no abnormalities were noted at necropsy and all animals showed expected gains in body weight.

- No signs of systemic toxicity were noted. Red colored staining of the feces was noted in all animals from Day 1 and up to 6 days after dosing.

The acute oral median lethal dose (LD₅₀) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight (Globally Harmonized Classification System-Unclassified).

In the acute dermal test, five males and five femaleswas given a single, 24 hour, semi-occluded dermal application to the intact skin at a dose level of 2000 mg/kg body weight.

- No mortality occured during the test, no abnormalities were noted at necropsy 

- No signs of systemic toxicity were noted. 

- Pink colored staining was noted at the test sites of all animals during the study and prevented accurate evaluation of erythema at the test sites of all animals 1 and 2 days after dosing. Signs of dermal irritation noted were hemorrhage of the dermal capillaries, small superficial scattered scabs, glossy skin and/or scab lifting to reveal glossy skin.

Regarding the body weight, three females showed body weight loss during the first week with expected gain in body weight during the second week. The remaining animals showed expected gains in body weight over the study period. 

The acute dermal median lethal dose (LD₅₀) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

Justification for selection of acute toxicity – oral endpoint
Only this study is available.

Justification for selection of acute toxicity – dermal endpoint
Only this study is available.

Justification for classification or non-classification

Based on the oral and dermal LD50 determined, no classification is required.

- oral toxicity:

Based on the above stated assessment of the acute oral toxicity thesubstance does need to be classified as Acute Oral toxicity Category 4 according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and accordingCLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.

- dermal toxicity:

Based on the above stated assessment of the acute dermal toxicity of the substance, the substance does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and accordingCLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council)as implementation of UN-GHS in the EU.