Xanthylium, 3,6-bis(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-, molybdatetungstatephosphate

Endpoint summary

Description of key information

The acute oral median lethal dose (LD 50) of the test item in the female Wistar strain rat was estimated to be >2000 mg/kg body weight (Globally Harmonized Classification System- Category 5). Category 5 has not been adopted by the EU according to Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

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.  The test item is not classified in accordance with GHS.

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:

Experimental Start Date (animal arrival): 05 October 2021
Experimental Completion Date: 03 November 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

Chemical name: Xanthylium, 3,6-bis(ethylamino)-9[2(methoxycarbonyl) phenyl]-2,7 dimethyl-,molybdatetungstatephosphate
CAS number: 85959-61-1
Lot number: 14026,
Magenta powder.
Received: 15 September 2021
Storage 15-25°C, protected from light.
Purity: 97-100%
Expiry date: 01 March 2022.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Species Selection
The rat has been selected because it is the rodent species preferred by various regulatory authorities. Background data are available. Females are used because, in literature surveys of conventional LD50 tests where a difference between the sexes has been seen, females have been shown to be slightly more sensitive than males.

Animal Specifications and Acclimatisation
Female (nulliparous, non-pregnant) Crl: WI(Han) strain rats were obtained from Charles River (UK) Ltd., Margate. All animals were given a clinical inspection for ill health on arrival and a sample was weighed.

Rats were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages using a sequence that reduced selective bias. The condition of the animals was assessed daily throughout the acclimatisation period of 7 to 15 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups at least one day prior to dosing.
The rats were in a body weight range of 150 to 183 g on Day 1. Based on information from the supplier the rats were approximately 8 to 10 weeks old on Day 1. The weight variation did not exceed ±20% of the mean weight.

Environmental Conditions, Diet, Water and Bedding
The animals were kept in the following conditions except for short periods of time where experimental procedures dictated otherwise.

Housing
The animals were housed in groups of up to six in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).

Bedding was provided on a weekly basis to each cage by use of clean European soft wood bedding (Datesand Ltd., Manchester, UK).
Each batch of bedding was analysed for specific constituents and contaminants. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Water
Mains water was provided ad libitum via water bottles. The water was periodically analysed for specific contaminants.
No contaminants were present in water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Diet
Throughout the study the animals had access to 5LF2 EU Rodent Diet 14%, which was freely available to the animals at all times, except for a period of fasting from the evening of the day prior to dosing (Day-1) until approximately 3 hours after dosing. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
No contaminants were present in diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Environment
The animal rooms were designed to permit a minimum of 15 air changes per hour. The target temperature and humidity ranges were 19 to 25°C and 40 to 70% respectively (see Protocol Deviation, Section 8). Daily recordings of maximum and minimum temperature and humidity were made.

Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.

Environmental Enrichment
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.
Environmental enrichment materials were removed during the period of fasting.

Animal Identification
A number written on the tail in indelible ink on the day before dosing individually identified the rats. A colour-coded card on each cage gave information including study number, group number, animal numbers and sex.

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

The test article was dispersed in 1% w/v aqueous methyl cellulose.

Details on oral exposure:

Doses were administered orally, by gavage, using plastic syringes and rubber catheters. Each rat was dosed once on Day 1, by passing the tip of a catheter along the oesophagus and instilling the test article into the gastric lumen.

Individual doses (mL) were calculated using the fasted body weights of the rats on the morning of dosing (Day 1) and the dose volume of 10 mL/kg.


Test Article Formulation
Dose levels were expressed gravimetrically and in terms of test article received (without regard to purity or active content). The test article was dispersed in 1% w/v aqueous methyl cellulose because the test article did not dissolve / suspend in purified water. The formulated concentrations were calculated from the selected dose level and the dose volume of 10 mL/kg. All formulations were used within two hours of preparation.
Dose containers were repeatedly inverted prior to administration to ensure homogeneity.

Doses:

Dose Level: 2000 (mg/kg)
Dose Concentration: 200 (mg/L)
Dose Volume: 10 (mL/kg)

No. of animals per sex per dose:

4 females

Control animals:

no

Details on study design:

Health Monitoring
All animals were observed at the beginning and the end of the working day for signs of ill health or overt toxicity.

Clinical Signs
Treated rats were observed closely for clinical signs of reaction to treatment. Clinical signs were recorded immediately post-dose, at approximately 15 and 30 minutes post dose, hourly between 1 and 4 hours post-dose (inclusive), twice daily on Days 2, 3 and 4 and once daily from the fifth to last day of the observation period. Individual records of clinical signs were maintained for each treated rat.

Body Weights
Rats were weighed on Day-1 (day before dosing) and on Days 1, 4, 8 and 15.

Terminal Procedures
Necropsy
Rats were killed on Day 15. Each animal was given isoflurane anaesthesia. Once a suitable deep plane of anaesthesia had been established, the animal was exsanguinated by the severing of major blood vessels. After exsanguination a full macroscopic necropsy was performed and all lesions were recorded.

The necropsy procedure included inspection of external surfaces and orifices, all viscera and tissue within the abdominal, thoracic and cranial cavities, free-hand sectioning of the liver and kidneys and examination of representative sections of mucosal surfaces of the stomach, small and large intestines.
No tissue preservation or histopathological assessment of tissues was undertaken.

Preliminary study:

Please see 'Any other information on results incl. tables' for this information.

Key result

Sex:

female

Dose descriptor:

other: Globally Harmonized System of Classification and Labelling of Chemicals (GHS)

Based on:

test mat.

Remarks on result:

other: Classified as Category 5 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS)

Mortality:

There were no deaths.

Clinical signs:

other: Piloerection was noted in the animal treated at 300 mg/kg, from 2 hours after dosing up to 3 hours after dosing. No clinical signs were seen in the animals treated at 2000 mg/kg, with the exception of pinks tails which were noted in four animals. This cli

Gross pathology:

No abnormalities were noted at necropsy.

Sighting Study

A preliminary test was performed to establish a dosing regimen for the main test. Dosing was as follows:

Dose Level (mg/kg)	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of Rats
300	30	10	1
2000	200	10	1

 

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The test article, C.I. Pigment Red 81:4, was classified as Category 5 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and was not classified in respect of its acute oral toxicity according to Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

Executive summary:

Objective

The objective of the study was to assess the acute toxicity of the test article, C.I. Pigment Red 81:4, following a single oral administration to the rat.

Introduction

Determination of the acute oral toxicity of a particular chemical product is among the first investigations carried out to assess the potential hazard it presents to manufacturer and consumer populations. It provides information on the likely consequences to man of short-term exposure to chemical products by ingestion and is a common basis for classification and labelling.

Results-

There were no deaths.

Piloerection was noted in the animal treated at 300 mg/kg, from 2 hours after dosing up to 3 hours after dosing.

No clinical signs were seen in the animals treated at 2000 mg/kg, with the exception of pinks tails which were noted in four animals. This clinical sign developed from Day 7 and lasted up to Day 15.

All rats achieved body weight gains during the first and second weeks of the study.

No abnormalities were noted at necropsy.

Conclusion-

The test article, C.I. Pigment Red 81:4, was classified as Category 5 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and was not classified in respect of its acute oral toxicity according to Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

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:

Testing was conducted between the 24th january 2018 and the 7th 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)

Deviations:

yes

Remarks:

Please see principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Please see principles of method if other than guideline.

Principles of method if other than guideline:

The following deviation from the Study Plan occurred: Based on current GLP guidance, at the time of issue/approval, the Study Plan did not contain sufficient information with regards test item identification. The required information is however included in the final study report. This deviation was considered not to have affected the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Specific details on test material used for the study:

Identification: Pigment Red 81:4
Batch: 64078
Purity: not supplied
Physical state/Appearance: red powder
Expiry Date: 30 June 2018
0Storage Conditions: room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS :
Five male and five 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 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 the previously dosed animals.

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 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 could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS :
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.

Type of coverage:

semiocclusive

Vehicle:

arachis oil

Remarks:

Moistened with arachis oil

Details on dermal exposure:

TEST SITE:
- Area of exposure: Not stated in report
- % coverage: Approximately 10% of the total body surface
- Type of wrap if used: ) A piece of surgical gauze was placed over the treatment area and semi occluded with a piece of self adhesive bandage.

REMOVAL OF TEST SUBSTANCE:
After the 24 Hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item.

TEST MATERIAL:
The appropriate amount of test item, moistened with arachis oil BP, 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.

VEHICLE:
Test item was moistened with Arachis oil BP.

Duration of exposure:

24 hours

Doses:

2000 mg/kg

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days.
- Frequency of observations and weighing: 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 application of the test item on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: 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.
- Other examinations performed: Clinical signs and body weight.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

no indication of skin irritation up to the relevant limit dose level

Mortality:

There were no deaths within the study.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period.

Gross pathology:

No abnormalities were noted at necropsy.

Table 1:   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

Table 2: 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	?s	?s	?s	?s	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1 Male	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2 Male	Erythema	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3 Male	Erythema	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4 Male	Erythema	?s	?s	?s	?s	?s	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	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0    = No reactions

?s = Test site stained red and precluded the accurate evaluation of erythema

Table 3     Individual Dermal Reactions - Females

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	2-0 Female	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-1 Female	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-2 Female	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-3 Female	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	2-4 Female	Erythema	?s	?s	?s	?s	?s	?s	?s	?s	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0   = No reactions

?s  = Test site stained red and precluded the accurate evaluation of erythema

Table 4     Individual Body Weights and Body Weight Changes:

Dose Level (mg/kg)	Animal Number and Sex	Body Weight (g) at Day			Body Weight Change (g) During Week
		0	7	14	1	2
2000	1-0 Male	252	280	298	28	18
	1-1 Male	244	278	302	34	24
	1-2 Male	261	280	295	19	15
	1-3 Male	239	254	280	15	26
	1-4 Male	242	260	283	18	23
	2-0 Female	217	220	230	3	10
	2-1 Female	227	232	241	5	9
	2-2 Female	214	216	237	2	21
	2-3 Female	227	230	239	3	9
	2-4 Female	217	230	249	13	19

 

Table 5     Individual Necropsy Findings:

Dose Level (mg/kg)	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	1-0 Male	Killed Day 14	No abnormalities detected
	1-1 Male	Killed Day 14	No abnormalities detected
	1-2 Male	Killed Day 14	No abnormalities detected
	1-3 Male	Killed Day 14	No abnormalities detected
	1-4 Male	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
	2-4 Female	Killed Day 14	No abnormalities detected

Interpretation of results:

GHS criteria not met

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. The test item is not classified in accordance with GHS.

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 undiluted 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: Red colored staining was noted onthe treated skin siteson Days 1 to 9 and prevented the macroscopic evaluation of erythema. No other skin reactions were noted.

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

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:

adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Acute Oral Toxicity:

Introduction

Determination of the acute oral toxicity of a particular chemical product is among the first investigations carried out to assess the potential hazard it presents to manufacturer and consumer populations. It provides information on the likely consequences to man of short-term exposure to chemical products by ingestion and is a common basis for classification and labelling.

Results-

There were no deaths.

Piloerection was noted in the animal treated at 300 mg/kg, from 2 hours after dosing up to 3 hours after dosing.

No clinical signs were seen in the animals treated at 2000 mg/kg, with the exception of pinks tails which were noted in four animals. This clinical sign developed from Day 7 and lasted up to Day 15.

All rats achieved body weight gains during the first and second weeks of the study.

No abnormalities were noted at necropsy.

Conclusion-

The test article, C.I. Pigment Red 81:4, was classified as Category 5 in respect of its acute oral toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and was not classified in respect of its acute oral toxicity according to Regulation (EC) No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

 

Justification for selection of acute toxicity – oral endpoint

The study was conducted on the test substance, in an appropriate test species and according to internationally recognised OECD guidelines. The study is considered to be of reliability 1.

 

Acute dermal toxicity:

Methods:

A group of ten animals (five males and five females) was given a single, 24 hour, semi‑occluded dermal application of the undiluted 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: Red colored staining was noted onthe treated skin siteson Days 1 to 9 and prevented the macroscopic evaluation of erythema. No other skin reactions were noted.

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

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.

 

 

Justification for selection of acute toxicity – dermal endpoint

The study was conducted on the test substance, in an appropriate test species and according to internationally recognised OECD guidelines. The study is considered to be of reliability 1.

 

Justification for classification or non-classification

The acute oral median lethal dose (LD ₅₀) of the test item in the female Wistar strain rat was estimated to be >2000 mg/kg body weight.

 

Category 5 Acute toxicity hazard categories has not been adopted by the CLP regulation (Regulation (EC) No 1907/2006) and is therefore not classified according to CLP.