Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25Sep2001 to 21Jan2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not applicable

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

EC Number:

269-503-2

EC Name:

Methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Cas Number:

68259-00-7

Molecular formula:

C14H16N3.CH3O4S

IUPAC Name:

methyl 1-methyl-4-[(methylphenylhydrazono)methyl]pyridinium sulphate

Test material form:

solid: particulate/powder

Details on test material:

See study reports for batch and purity information

Specific details on test material used for the study:

The test material, MIP 2982 (identified at receipt as MIP Yellow 2982; Batch No. 028400A8AA), was received from the Sponsor on September 10, 2001.

Test animals

Species:

rat

Strain:

other: Crl:CD® (SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
Young, adult male and female Crl:CD® (SD)IGS BR rats were received from Charles River Laboratories, a USDA-approved supplier located in Raleigh, North Carolina, on September 18 and 20, 2001 and October 02, 2001.

The animals were assigned temporary numbers during acclimation and identified by individual numbered ear tag and by cage label for the duration of the study.

Housing
The animals were pair housed during the acclimation period and singly housed during the study period in hanging, stainless-steel, wire-mesh cages measuring approximately 24.2 cm X 22.0 cm x 17.3 cm.

Diet
A commercial diet (PMI* Feeds, Inc. Certified Rodent Diet* #5002 - pellet form) was available ad libitum during the acclimation and study periods, with the exception of a period for 17-20 hours prior to and approximately 4 hours after test material administration. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. Results are on file at Covance-Vienna.

Water
Water, via an automatic watering system, was available ad libitum during the acclimation and study periods. The water was analyzed on a routine basis for specified microorganisms, pesticides, alkalinity, heavy metals, and halogens. Specified nutrient and contaminant analyses are on file at Covance - Vienna. There were no contaminants, known or reasonably anticipated, in the diet or water at levels that might interfere with the validity of this study.

Environment
The temperature and relative humidity in the animal room were monitored at least once daily. Room controls were set to maintain temperatures and relative humidity of 18-26°C (64-79° F) and 50%±20%, respectively. A 12-hour light/12-hour dark cycle was maintained in the room housing the animals. Ten or greater air changes per hour were maintained in the room housing the animals.

Acclimation
Before being considered for study use, the animals were acclimated to laboratory conditions. After at least 5 days of acclimation, a staff veterinarian deemed them to be healthy and free from disease and physical abnormalities, and then released the animals for use in the study.

Selection of Animals
Animals used in this study were allocated from all animals available for study that were within the protocol-specified weight range (200-300 g). After randomizing, using computer-generated random numbers, the rats were assigned into this study and to dose groups. The weight variation of the animals selected for the study did not exceed ±20% of the mean body weight of each sex.

Justification of Species Selection
The rat was selected for use on this study because, historically, rats have been used as a representative of a rodent species and are recommended by various regulatory agencies.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Dose Preparation
The test material was mixed with cell culture grade water and stored at room temperature until administration.

Method of Administration
All animals received a single dose of the test material administered by oral gavage. The individual dose volume was based on each animal's body weight taken just before dosing. Food was withheld from the animals for 17 to 20 hours before and approximately 4 hours after dosing.

Reason for Dosing Route
Potential human exposure is by the oral route.

Doses:

All animals received a single dose of the test material administered by oral gavage. The individual dose volume was based on each animal's body weight taken just before dosing.

No. of animals per sex per dose:

Limit test
500,1000 and 1500 mg/kg - 2 males and 2 females
2000 mg/kg – 5 males and 5 females

Oral Tox Study
1000 mg/kg - 5 males
500 mg/kg – 5 females

Control animals:

no

Details on study design:

OBSERVATION OF ANIMALS
Clinical Observations
The animals were observed twice daily (at least 4 hours apart) for mortality and moribundity. Immediately after dosing and at approximately 1, 2.5, and 4 hours after dose administration and once daily thereafter for at least 14 days, animals were observed in the cage for indications of toxicity or ill health. (See Protocol Deviation section for exceptions.) Any effects were recorded as they were observed, noting only those animals for which an observation was made.

Body Weights
The animals were weighed the day of (prior to) test material administration (following fasting), weekly thereafter, and at termination (fasted).

TERMINATION AND POSTMORTEM PROCEDURES
Scheduled Sacrifice
At termination of the experimental phase, surviving animals were fasted ovemight (Day 14), weighed, anesthetized with an appropriate barbiturate, and exsanguinated.

Necropsy
All animals (scheduled and nonscheduled deaths) were subjected to an abbreviated gross necropsy examination of the cervical, thoracic, and abdominal viscera, performed by trained personnel using procedures approved by board-certified pathologists. All abnormalities were recorded. After necropsy, the animals were discarded and no tissues were saved.

Statistics:

N/A

Results and discussion

Preliminary study:

RESULTS OF THE DOSE LIMIT TEST
Dosing Information. The animals used in the dose limit test were dosed on September 26 or October 02, 2001. At initiation of dosing the males were approximately 8 weeks old and the females were approximately 10 weeks old, with a weight range of 215 to 290 g and 202 to 230 g, respectively. At the termination of this assay, all surviving animals were euthanized with Euthanasia 6, followed by exsanguination, and necropsied.
Mortality
A summary of mortality is presented in Table 1. All males and females dosed at 500 mg/kg, all males dosed at 1000 mg/kg, and one male dosed at 1500 mg/kg survived until study termination. All females dosed at 1000, 1500, and 2000 mg/kg died by Day 1. One male dosed at 1500 mg/kg died on Day 0 and all males dosed at 2000 mg/kg died by Day 1.
Clinical Signs
Individual clinical observations are presented in Table 2. The animals that survived to study termination were noted with soft faeces, slight hypoactivity, faecal and urine staining, discoloured urine and/or faeces, few faeces, and/or stained hair in the genital area (this finding persisted until the study termination). All remaining animals were noted with several of these findings prior to death.
Body Weights
Individual and mean body weights and body weight changes are presented in Table 3. With the exception of a 11-g weight loss (Days 0 to 14) in one male dosed at 1000 mg/kg and a 30-g loss (Days 0-7) in one male at 1500 mg/kg, all animals that survived to the study termination gained weight during the course of the study.
Necropsy
Individual necropsy findings are presented in Table 4. With the exception of one 1500 mg/kg male with an enlarged heart, no visible lesions were noted in the animals that survived to study termination. Macroscopic findings in the remaining animals usually involved the stomach, cecum, ileum, duodenum, jejunum, colon, and/or bladder. Findings noted were discoloured and/or distended organ filled with yellow fluid.

Effect levels

Key result

Sex:

male/female

Dose descriptor:

approximate LD50

Effect level:

1 000 mg/kg bw

Based on:

test mat.

Mortality:

Two of the five males in the 1000 mg/kg dose group died on Day 0 and Day 2. All remaining animals survived until the scheduled sacrifice.

Clinical signs:

other: Prior to the death of the two males in the 500 mg/kg dose group, hypoactivity, ataxia, liquid or mucoid faeces, discoloured faeces, and/or discoloured urine were noted. In the remaining 500 mg/kg males and the females dosed at 250 mg/kg, hypoactivity, ata

Gross pathology:

With the exception of a pale area in the liver of one female, no visible lesions were noted in the animals that survived to study termination. Macroscopic findings in the two males that died involved the stomach, ileum, duodenum, jejunum, and colon (distended organ yellow fluid), and the bladder (moderately distended and filled with yellow lumen fluid containing a hard, yellow material) of one animal.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The test item was toxic by the oral route at dose levels of 1000 mg/kg in the males and 500 mg/kg in the females under the conditions of this study. The LD50 was estimated to be 1000 mg/kg. The test item should therefore be classified as an actute toxicity Category 4 by oral exposure according to EC 1272/2008.

Executive summary:

This study was designed to assess the acute oral toxicity produced when the test material was administered by oral gavage to male and female CD rats.

 

A dose limit test was performed at the below concentrations using both male and female CD rats.

500,1000 and 1500 mg/kg - 2 males and 2 females

2000 mg/kg – 5 males and 5 females

 

Based on mortality and clinical observations drung the limit test the following dose levels were chosen for the oral toxicity study.

1000 mg/kg - 5 males

500 mg/kg – 5 females

 

Two of the five males in the 1000 mg/kg dose group died on either Day 0 or Day 2 following signs that included hypoactivity, ataxia, squinted eyes, liquid or mucoid faeces, discoloured faeces and/or discoloured urine. Macroscopic findings at necropsy included the stomach, ileum, duodenum, jejunum, and colon (distended organ filled with yellow fluid), and the bladder (moderately distended and filled with yellow lumen fluid containing a hard, yellow material) of one animal.

 

In the remaining males dosed at 1000 mg/kg and the females dosed at 500 mg/kg, hypoactivity, ataxia, squinted eyes, liquid or mucoid faeces, discoloured faeces, discoloured urine, urine and/or faecal staining, crust around the eyes and/or discoloured hair in the genital region were noted. The animals gained weight during the course of the study. With the exception of a pale area in the liver of one female, no visible lesions were noted in the animals that survived to study termination.

 

The test item was toxic by the oral route at dose levels of 1000 mg/kg in the males and 500 mg/kg in the females under the conditions of this study. The LD50 was estimated to be 1000 mg/kg. The test item should therefore be classified as an acute toxicity Category 4 by oral exposure according to EC 1272/2008.