Multi constituent substance

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD guideline 408 in compliance to GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Reference 2

Reference Type:

other: Published secondary source

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Multi constituent substance

EC Number:

916-603-5

IUPAC Name:

Multi constituent substance

Details on test material:

Test substance : Basic Red 76 (COLIPA number C008)
Batch number : 0057891101
Methylsulphate anion : 15.9%
Chloride ion : 2.7%
Water : 4.1%
o-Anisidine : 19ppm
Purity : 98.6% (HPLC)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The test substance and vehicle control were administered once daily by oral gavage for 91 consecutive days.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
60 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
250 mg/kg bw
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Twelve/sex/dose group. Five/sex/dose group for recovery (4 weeks)

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance was administered as an aqueous solution in a single dose by gavage for 91 consecutive days in daily doses of 60, 250 and 1000 mg/kg bw/day based on the results of the dose range finding study of the prenatal developmental toxicity study (0, 200, 600, 1000 mg/kg bw/day). The control animals received the vehicle alone. During the study mortality, signs of toxicity, body weight and food consumption were recorded. Functional observation tests were performed in week 12. Ophthalmoscopy was performed before the start of treatment and in week 13. The animals of the recovery groups were additionally examined during the 4 week treatment free period. At the end of the study the animals were sacrificed and subjected to pathological investigations.

Results and discussion

Results of examinations

Details on results:

No mortality occurred that was considered to be due to the toxicity of the test item. One female of the 60 mg/kg bw/day dose group was found dead on day 60. Based on microscopic and macroscopic findings the cause of death was considered to be related to the dosing procedure. All other animals survived the duration of the study.

Red staining of various body parts and red discolouration of faeces and urine observed among the dose groups were considered to be related to the staining properties of the test item and/or a metabolite and is considered to be of no toxicological significance.

Clonic spasms observed in some animals of all dose groups after treatment including the control group in the last few weeks of treatment were considered not to be significant in toxicological terms. This observation was of an intermittent and infrequent nature and was not supported by abnormalities during functional observation testing.

No toxicologically relevant changes in body weight and food intake quantities were observed and no abnormalities were seen during ophthalmoscopic examination.

At 250 and 1000 mg/kg bw/day effects on haematology parameters were indicative of destruction of red blood cells and included increased tissue iron storage in the spleen and liver and increased serum bilirubin levels. Elevated numbers of reticulocytes, increased severity of extramedullary haemopoiesis in the spleen and erythropoiesis in the sternal bone marrow indicate a regenerative response to the lower red blood cell counts. Congestion of the spleen and increased spleen weights (with enlargement) were related to these processes. Increased methaemoglobin formation at 250 and 1000 mg/kg bw/day indicates that the test item may convert haem to the ferric state. The increased incidence/severity of hemosiderin pigment in the spleen at 1000 mg/kg bw/day at the end of the recovery period may indicate a residual presence of tissue iron storage. Based on normal red blood cell counts and the absence of extramedullary haematopoiesis it was considered that haematological effects had widely resolved during the 4 week recovery period.

Thyroid follicular hypertrophy in combination with hypertrophy of the adenohypophyseal cells of the pituitary as observed at the 1000 mg/kg bw/day dose level probably reflect a perturbation of thyroid-pituitary hormone homeostasis.

At 250 and 1000 mg/kg bw/day there was clear evidence of an adverse effect of the test item on red blood cell life span as indicated by evidence for haemolysis and increased methaemoglobin levels at these dose levels. At 60 mg/kg bw/day evidence for an effect on red blood cell turn over was marginal in nature and occurred in the absence of any evidence of red blood cell destruction or increased extramedullary haematopoiesis and was considered not to reflect an adverse effect on red blood cell turn over.

Effect levels

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a subchronic toxicity study a NOAEL of 60 mg/kg bw/day was determined following treatment by oral gavage in rats.