Hydrogen 9-amino-7-phenyl-5-(phenylamino)-4,10-disulphonatobenzo[a]phenazinium, disodium salt

Endpoint summary

Description of key information

A group of three fasted females was treated with the test item at a dose level of 2112 mg/kg body weight (equivalent to 2000 mg active ingredient/kg body weight). This was followed by a further group of three fasted females at the same dose level. Dosing was performed sequentially. The test item was administered orally as a suspension in distilled water. During the 14 day observation period, clinical signs and body weight development were monitored. Thereafter all animals were subjected to gross necropsy.

Results: There were no deaths. There were no signs of systemic toxicity. Black staining of the feces was noted in the cage of the second group of animals. All animals showed expected gains in body weight. 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 2112 mg/kg body weight (equivalent to 2000 mg active ingredient/kg body weight) (Globally Harmonized Classification System - Unclassified).

The test item Acid Violet 50 was evaluated for Acute Dermal Toxicity in Sprague Dawley Rats as per the OECD guideline for the testing of chemicals No. 402, “Acute Dermal Toxicity - Fixed Dose Procedure”.

The animals were dosed in a stepwise procedure with one female at a time in range finding study. A starting dose of 2000 mg/kg body weight was selected.

No clinical signs and mortalities were observed at the dose level of2000 mg/kg body weight in range finding study. Hence, during main study, two animals were administered with the same dose level of 2000 mg/kg body weight. No clinical signs and mortalities were observed at the dose level of 2000 mg/kg body weight. Hence, no further testing was carried out.

No mortality, clinical signs and skin reactions were noted. No treatment related changes in body weight and percent change in body weight with respect to day 1 were noted. Normal increase in body weights were noted during the observation period. No treatment related gross pathological changes were noted at 2000 mg/kg body weight during necropsy.

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Animal Information
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, 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 mean body weight of any previously dosed animals.
Animal Care and Husbandry
The animals were housed in groups three 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 Envigo RMS (UK) Limited, 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 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.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

All animals were dosed once only by gavage

Doses:

2112 mg/kg (equivalent to 2000 mg active ingredient/kg)

No. of animals per sex per dose:

3 females per dose, 2 dose groups

Control animals:

no

Details on study design:

Using available information on the toxicity of the test item, 2112 mg/kg (equivalent to 2000 mg active ingredient/kg body weight) 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)
2112*, 211.2, 10, 3
2112*, 211.2, 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 14 days.
Individual body weights were recorded prior to dosing and 7 and 14 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.

Preliminary study:

Not applicable.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

act. ingr.

Remarks on result:

other: 95% CL not reported

Mortality:

Individual mortality data are given in Appendix 1.
There were no deaths.

Clinical signs:

other: Individual clinical observations are given in Appendix 1. No signs of systemic toxicity were noted during the observation period. Black staining of the feces was noted in the cage of the second group of animals.

Gross pathology:

Individual necropsy findings are given in Appendix 3.
No abnormalities were noted at necropsy.

Other findings:

None

Appendix1     Individual Clinical Observations and Mortality Data

		Effects Noted After		Dosing (Hours)		Effects Noted During			Period After		Dosing (Days)
Dose Level mg/kg	Animal Number and Sex	½	1	2	4	1F	2F		3F	4	5	6	7	8	9		10		11		12	13	14
2112*	1-0 Female	0	0	0	0	0	0		0	0	0	0	0	0	0		0		0		0	0	0
	1-1 Female	0	0	0	0	0	0		0	0	0	0	0	0	0		0		0		0	0	0
	1-2 Female	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

*=    Equivalent to 2000 mg active ingredient/kg body weight

0=   No signs of systemic toxicity

F =   Black staining of the feces in the cages of animal numbers 2-0, 2-1 and 2-2

Appendix2     Individual Body Weights and Body Weight Changes

		Body Weight (g) at Day			Body Weight Gain (g) During Week
Dose Level mg/kg	Animal Number and Sex	0	7	14	1		2
2112*	1-0 Female	145	173	186	28		13
	1-1 Female	148	163	176	15		13
	1-2 Female	143	162	173	19		11
	2-0 Female	176	194	209	18		15
	2-1 Female	161	180	199	19		19
	2-2 Female	164	188	204	24		16

*=    Equivalent to 2000 mg active ingredient/kg body weight

Appendix3     Individual Necropsy Findings

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2112*	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
	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

*=    Equivalent to 2000 mg active ingredient/kg body weight

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2112 mg/kg body weight (equivalent to 2000 mg active ingredient/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

A group of three fasted females was treated with the test item at a dose level of 2112 mg/kg body weight (equivalent to 2000 mg active ingredient/kg body weight). This was followed by a further group of three fasted females at the same dose level. Dosing was performed sequentially.

The test item was administered orally as a suspension in distilled water. During the 14 day observation period, clinical signs and body weight development were monitored. Thereafter all animals were subjected to gross necropsy.

Results

Mortality. There were no deaths.

Clinical Observations. There were no signs of systemic toxicity. Black staining of the feces was noted in the cage of the second group of animals.

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 2112 mg/kg body weight (equivalent to 2000 mg active ingredient/kg body weight) (Globally Harmonized Classification System - Unclassified).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

20 December 2017 to 02 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

OECD Guideline for Testing of Chemicals No. 402 (Section 4: Health Effects) “Acute Dermal Toxicity: Fixed Dose Procedure” adopted on 09 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks
- Weight at study initiation: 220.21 g to 232.16 g
- Fasting period before study: No
- Housing: L 430 x B 285 x H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through Reverse osmosis unit
- Acclimation period: Start: 20 December 2017 End: 01 January 2018

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°C to 23.0°C
- Humidity (%): 48% to 67%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 20 December 2017 To: 11 January 2018

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: approximately 10% of the total body surface
- % coverage: approximately 10% of the total body surface
- Type of wrap if used: non-irritating adhesive tape

REMOVAL OF TEST SUBSTANCE
- Washing (if done): distilled water
- Time after start of exposure: 24 hours

TEST MATERIAL
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied (volume): 0.5 mL

Duration of exposure:

The contact period of test item was at least 24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

range finding study: 1 female (2000 mg/kg body weight)
main study: 2 females (2000 mg/kg body weight)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: once daily for clinical signs of toxicity and twice daily for mortality and body weights weekly once
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Key result

Sex:

female

Dose descriptor:

LD0

Effect level:

>= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities observed

Clinical signs:

other: No treatment related clinical signs were observed

Gross pathology:

No treatment related gross pathological changes were observed

Phase of the Experiment

Dose (mg/kg body weight)

Animal No.

Sex

Time of Dosing (AM)

Clinical Signs of Toxicity and Mortality on Day 1

Clinical Signs of Toxicity and Mortality on days

20-30 mins

1 hr (±10 mins)

2 hrs (±10 mins)

4 hrs (±10 mins)

6 hrs (±10 mins)

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Range Finding Study

2000

Rc6201

F

11:14

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Main Study

2000

Rc6202

F

11:16

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

Rc6203

F

11:18

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

    N: Normal; F: Female; min: minutes; hr/hrs: hour/hours

Phase of the Experiment	Dose (mg/kg body weight)	Animal No.	Sex	Body Weight (g) on Days				Percent Change in Body Weight with Respect to Day
				1	8	15		1 to 8	1 to 15
Range Finding Study	2000	Rc6201	F	242.41	265.76	297.45		9.63	22.71
Main Study	2000	Rc6202	F	232.78	261.75	290.45		12.45	24.77
		Rc6203	F	246.70	272.59	301.63		10.49	22.27
		Mean		239.74	267.17	296.04		11.47	23.52
		±SD		9.84	7.67	7.91		1.38	1.77

     F: Female; SD: Standard Deviation

Phase of the Experiment	Dose (mg/kg body weight)	Animal No.	Sex	Fate	Gross Pathology Findings
					External	Internal
Range finding Study	2000	Rc6201	F	TS	NAD	NAD
Main Study	2000	Rc6202	F	TS	NAD	NAD
		Rc6203	F	TS	NAD	NAD

 NAD: No Abnormality Detected; F: Female;TS: Terminal Sacrifice             

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Under the experimental conditions employed and based on the above results, it is concluded that the acute dermal median lethal dose (LD50) of test item Acid Violet 50 in Sprague Dawley rats is >2000 mg/kg body weight

Executive summary:

The test item Acid Violet 50 was evaluated for Acute Dermal Toxicity in Sprague Dawley Rats as per the OECD guideline for the testing of chemicals No. 402, “Acute Dermal Toxicity - Fixed Dose Procedure”.

The study was performed in two phases i.e. range finding study and main study. Range finding study was performed with one female rat and main study was performed with two female rats. On the day before the application of the test item, fur on the dorso-lateral area of the trunk of the animals was removed by clipping closely with an electric hair clipper and care was taken to avoid abrading the skin.

The required quantity of the test item was moistened with 0.5 mL of distilled water to form thin paste using glass rod and applied as uniform film over an area of approximately 10% of the total body surface. The test item was held on to the applied surface by covering with cotton gauze dressing and wrapped with non-irritating adhesive tape and finally the application site was wrapped using semi-occlusive crepe bandage. The contact period of test item was 24 hours. At the end of the contact period, the residual test item was washed using distilled water and dried with absorbent cotton.

The animals were dosed in a stepwise procedure with one female at a time in range finding study. Based on sponsor’s specification, a starting dose of 2000 mg/kg body weight was selected from the fixed dose levels of 50, 200, 1000 and 2000 mg/kg body weight. No clinical signs and mortalities were observed at the dose level of 2000 mg/kg body weight in range finding study. Hence, during main study, two animals were administered with the same dose level of 2000 mg/kg body weight. No clinical signs and mortalities were observed at the dose level of 2000 mg/kg body weight. Hence, no further testing was carried out.

All the animals were observed for clinical signs of toxicity and mortality at 20 to 30 min, 1 hr (±10 mins), 2 hrs (±10 mins), 4 hrs (±10 mins) and 6 hrs (±10 mins) post dosing on day 1 and thereafter once daily for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. The body weight was recorded on day 1 before test item application and on day 8 and 15. At the end of observation period, all the animals were sacrificed under carbon dioxide anaesthesia and subjected to necropsy and detailed gross pathological examination.

No mortality, clinical signs and skin reactions were noted. No treatment related changes in body weight and percent change in body weight with respect to day 1 were noted. Normal increase in body weights were noted during the observation period. No treatment related gross pathological changes were noted at 2000 mg/kg body weight (range finding study and main study) during necropsy

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

No classification

No adverse effects were observed after oral or dermal exposure to limit doses.