Reaction mass of N-(4-(bis(4-(methylamino)phenyl)methylene)cyclohexa-2,5-dienylidene)-N-methylmethanaminium acetate and [4-[[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methylene]cyclohexa-2,5-dien-1-ylidene]dimethylammonium acetate and [4-[bis[4-(dimethylamino)phenyl]methylene]-2,5-cyclohexadien-1-ylidene]dimethylammonium acetate

Endpoint summary

Description of key information

LD50 rat (oral)>300 and < 2000 mg/kg

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

Liquid dark blue-violet

Species:

rat

Strain:

other: RccHan™:WIST albino

Sex:

female

Details on test animals or test system and environmental conditions:

Healthy nulliparous and non-pregnant
The animals were allocated without conscious bias to cages within the treatment groups.
They were housed in groups of one or four rats of the same sex.
Each animal was identified uniquely within the study by tail marking. Each cage label was color-coded and was identified uniquely with the study number, dose level and animal mark.
The animals were allowed to acclimatize to the conditions described below for at least five days before treatment. For those animals selected for this study, their body weights were in the range 160 to 181 g and they were approximately eight to twelve weeks of age prior to dosing (Day 1). The body weight variation did not exceed  20% of the mean body weight of any previously treated animals.

Animals were housed inside a limited access rodent facility (Building F21, Room 044/045). The facility was designed and operated to minimize the entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 20 to 24C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study.
Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.
Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.

The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fiber bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Teklad 2014C Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.

Each cage of animals was provided with Aspen chew blocks or balls for environmental enrichment. Chew blocks or balls were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
Each batch of diet was analyzed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinized and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier.
Certificates of analysis were received routinely from the supplier of the chew blocks or balls.
Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they are not presented.
No other specific contaminants that were likely to have been present in the diet or water were analyzed, as none that may have interfered with or prejudiced the outcome of the study was known.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test item was formulated at concentrations of 30 and 200 mg/mL in the vehicle, by adding the vehicle to the item and mixing. The formulation was administered at a volume of 10 mL/kg body weight

Doses:

dose range finding: 300 and 2000 mg/kg (In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose)
main study: 300 mg/kg

No. of animals per sex per dose:

dose range finding: 1 female per dose
main study: 4 females at the selected dose

Control animals:

no

Details on study design:

The appropriate dose volume of the test item was administered to each rat by oral gavage using a plastic syringe and plastic catheter.
Formulations were stirred before and throughout the dosing procedure

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

On Day 6 female number 22 dosed in the sighting study at 2000 mg/kg was sacrificed on humane grounds due to the severity of the clinical signs seen.

Clinical signs:

other: Clinical signs throughout the six days before death of the female dosed at 2000 mg/kg comprised salivation, purple staining in saliva, mouth and front paws, piloerection, partially closed eyelids, abnormally cold to touch, hunched posture, dark feces, dec

Gross pathology:

For the female that died at 2000 mg/kg macroscopic examination of the animal revealed congestion (characterized by darkened tissues/organs) of the subcutaneous tissue, heart, lungs and bronchi, liver, spleen and kidneys. Also, a small heart and spleen and enlarged, swollen or thickened tissues of the stomach were noted. A dark purple staining was observed in the stomach as well as purple fluid contents and dark purple staining in the duodenum, cecum and small and large intestines.

No abnormalities were noted in any surviving animal at the macroscopic examination at study termination on Day 15.

Conclusions:

The substance was tested for acute oral toxicity following OECD 420. Under the experimental conditions the LD50 > 300 and < 2000 mg/kg.

Executive summary:

The substance was tested for acute oral toxicity following OECD 420 (rats). Fasted female rats received a single oral gavage dose of the test item, formulated in purified water, at the following dose levels: Sighting investigations: 300 and 2000 mg/kg body weight

Main study: Based on the results of the sighting investigations a further four fasted females were similarly dosed at 300 mg/kg body weight.

During the study, clinical condition, body weight and macropathology investigations were undertaken. Under the experimental conditions the LD50 > 300 and < 2000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008), table 3.1.1, Acute toxicity hazard categories and acute toxicity estimates (ATE) defining the respective categories:

For Acute toxicity oral route:

Category 1: ATE <= 5 mg/kg bw

Category 2: 5 < ATE <= 50 mg/kg bw

Category 3: 50 < ATE <= 300 mg/kg bw

Category 4: 300 < ATE <= 2000 mg/kg bw

The LD50 of the test substance was determined to be >300 and < 2000 mg/kg bw, therefore, the test substance is classified as category 4, H302 Acute toxicity by oral exposure.