9-[bis(2-hydroxyethyl)amino]-5-[(3-methylphenyl)amino]-7-phenylbenzo[a]phenazin-7-ium chloride

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

EC Number:

941-946-2

Molecular formula:

C33H31ClN4O2

IUPAC Name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

Details on test material:

purity; 89.7 % (w/w) (main component)

Test animals

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 five 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 eight to twelve weeks of age. The body weight variation did not exceed ±20% of the mean body weight of the initially dosed group.

The animals were housed in groups of three in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four 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 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

All animals were dosed once only by gavage

Doses:

300 mg/kg
2000 mg/kg

No. of animals per sex per dose:

3 animals at 300 mg/kg
6 animals at 2000 mg/kg

Control animals:

no

Details on study design:

In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.

Groups of fasted animals were treated as follows:

Dose Level(mg/kg) / Concentration(mg/mL) / Dose Volume(mL/kg) / Number of Rats (Female)
300 / 30 / 10 / 3
2000 / 200 / 10 / 3
2000 / 200 / 10 / 3

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 group to confirm the survival of the previously dosed animals.

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 fourteen days.

Individual body weights were recorded prior to dosing and seven and fourteen days after treatment.

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

Statistics:

None recorded.

Results and discussion

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 95% CL not reported

Mortality:

Individual mortality data are given in Table 1.

There were no deaths.

Clinical signs:

other: Individual clinical observations are given in Table 2 and Table 3. No signs of systemic toxicity were noted during the observation period. Black stained feces was noted in all animals during the study.

Gross pathology:

Individual necropsy findings are given in Table 6 and Table 7.

No abnormalities were noted at necropsy.

Other findings:

None

Any other information on results incl. tables

Table 1     Mortality Data

Dose Level mg/kg	Sex	Number of Animals Treated	Deaths During Day of Dosing (Hours)				Deaths During Period After Dosing (Days)								Deaths
			½	1	2	4	1	2	3	4	5	6	7	8-14
300	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3
2000	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3
	Female	3	0	0	0	0	0	0	0	0	0	0	0	0	0/3

Table 2     Individual Clinical Observations - 300 mg/kg

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
300	1-0 Female	0	0	0	0	0F	0F	0F	0	0	0	0	0	0	0	0	0	0	0
	1-1 Female	0	0	0	0	0F	0F	0F	0	0	0	0	0	0	0	0	0	0	0
	1-2 Female	0	0	0	0	0F	0F	0F	0	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

F =   Black stained feces

Table 3     Individual Clinical Observations - 2000 mg/kg

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	2-0 Female	0	0	0	0	0F	0F	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	0	0	0	0	0F	0F	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	0	0	0	0	0F	0F	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	0	0	0	0F	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	3-1 Female	0	0	0	0F	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0
	3-2 Female	0	0	0	0F	0F	0F	0F	0F	0	0	0	0	0	0	0	0	0	0

0=   No signs of systemic toxicity

F =   Black stained feces

Table 4     Individual Body Weights and Body Weight Changes - 300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Body Weight (g) at Day			Body Weight Gain (g) During Week
		0	7	14	1	2
300	1-0 Female	166	178	190	12	12
	1-1 Female	162	170	183	8	13
	1-2 Female	146	154	179	8	25

Table 5     Individual Body Weights and Body Weight Changes - 2000 mg/kg

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	2-0 Female	162	178	203	16	25
	2-1 Female	152	173	194	21	21
	2-2 Female	163	179	193	16	14
	3-0 Female	148	169	182	21	13
	3-1 Female	168	185	201	17	16
	3-2 Female	155	170	186	15	16

Table 6     Individual Necropsy Findings - 300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
300	1-0 Female	Killed Day 14	No abnormalities detected
	1-1 Female	Killed Day 14	No abnormalities detected
	1-2 Female	Killed Day 14	No abnormalities detected

Table 7     Individual Necropsy Findings - 2000 mg/kg

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

Applicant's summary and conclusion

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 2500 mg/kg body weight (Globally Harmonized Classification System - Unclassified). The LD50 cut-off (mg/kg body weight) was determined to be: Infinity.

According to the findings in this study Duasyn Blue T48 gefriergetrocknet does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.

Executive summary:

Introduction

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

 

 

Methods

A group of three fasted females was treated with the test item at a dose level of 300 mg/kg body weight. Based on the results from this dose level, further groups of fasted females were treated at a dose level of 2000 mg/kg body weight. Dosing was performed sequentially.

 

The test item was administered orally as a suspension in distilled water. 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. All animals showed black stained feces during the study.

 

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 2500 mg/kg body weight (Globally Harmonized Classification System - Unclassified). The LD50 cut-off (mg/kg body weight) was determined to be: Infinity.

 

According to the findings in this study the test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16 December 2008.