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

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

289-056-7

EC Name:

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

Cas Number:

85959-61-1

Molecular formula:

C162H180N12O18Cl6+O3.P2O5.xWO3.yMoO3

IUPAC Name:

heptakis(N-[(3E)-6-(ethylamino)-9-[2-(methoxycarbonyl)phenyl]-2,7-dimethyl-3H-xanthen-3-ylidene]ethan-1-aminium) dioxomolybdenumbis(olate) dioxotungstenbis(olate) phosphate

Test material form:

other: granular solid

Details on test material:

Identification: Pigement Red 81:4
Appearance/Physical State: Magenta colored granula solid
Batch: S-3101
Expiry Date: 01 June 2014
Storage Conditions: Room temperature in the dark

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.

Test animals

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.

Administration / exposure

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.

Results and discussion

Preliminary study:

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

Effect levels

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.