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:

2014

Report date:

2014

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:

916-604-0

IUPAC Name:

Multi constituent substance

Details on test material:

Test substance : Basic Brown 17 (COLIPA number B007)
Batch number : 64960101
Methylsulphate anion : 11.6%
Chloride ion : 3.3%
Water : 6.5%
Basic Red 118 : 4.5%
Purity : 94.2% (HPLC)

Test animals

Species:

rat

Strain:

other: Wistar Hannover

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/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
120 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
180 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Results and discussion

Results of examinations

Details on results:

Two control males, two males and one female treated with a test item dose of 180 mg/kg bw/day were found dead during the treatment period. Body weight and food consumption were unaffected by the treatment. No ophthalmological changes occurred during the treatment. During the daily before and after treatment observations no clinical signs were noted. No significant differences were observed between the control and test item treated animals for the weekly clinical signs and functional observation testing. No changes in motor activity were observed in animals receiving the test item compared with controls. There were no significant haematological changes noted during the study. Two mid dose females, three females and three males of the high dose showed moderate to marked alteration in aspartate aminotransferase. Since only one of the high dose females also had a raised level of alanine aminotransferase liver inquiry was excluded. In the high dose group significantly raised gamma glutamyl transferase, cholesterol, triglycerides, glucose levels and lowered sodium levels were observed. At the end of the recovery phase these parameters were similar to the control values. Some of the high dose animals showed bilirubinuria but as no hyperbilirubinaemia was observed it was thought that this could be related to the test item and/or test item metabolites in the urine.

Post mortem investigations of the high dose rats showed dark colouration in the brain, heart, kidneys, ovaries, skeletal muscle, spleen and thyroid with the females more effected than males. In the mid dose both sexes showed dark colouration of the spleen and thyroid. Some females also had dark colouration in the heart and skeletal muscle. Histopathology showed yellow/brown pigmentation was the most relevant treatment related change observed in the heart, kidneys, liver, spleen, thyroids ileum and skeletal muscle of animals of both sexes treated with a dose of >= 120 mg/kg bw/day when compared with controls. Animals dosed at 180 mg/kg bw/day showed yellow/brown pigmentation in the adrenals, ovaries, uterus, mesenteric/cervical lymph nodes and thymus. An increased incidence of extramedullary haemopoiesis in the spleen in all treated groups was recorded. The remaining lesions including the statistically significant increased incidence of inflammatory cell foci noted in the heart of males dosed at 60 mg/kg bw/day were considered either incidental in origin or an expression of spontaneous pathology commonly seen in this species under the experimental conditions used.

Post mortem examination showed dark colouration was still present after 4 weeks of recovery in the brain, heart, skeletal muscle, spleen and thyroid of treated males and females when compared with the controls. Histopathology of the recovery group showed yellow/brown pigmentation was still present in the adrenals, heart, kidneys, liver, ovaries, spleen, thyroid, skeletal muscle and mesenteric/cervical lymph nodes in the high dose group. Absolute and relative thyroid weights were lower than controls in females dosed at 180 mg/kg bw/day at the end of the recovery period. In the same animals the relative liver weights were higher than controls. Changes in clinical chemistry parameters that could be correlated with the histopathological changes detected in the heart and skeletal muscle were a moderate to marked increase in aspartate aminotransferase detected in some animals dosed at 120 and 180 mg/kg bw/day at the end of the treatment period.

Post mortem examination of both the control males and the high dose female which were found dead showed incomplete collapse in the lungs. Both the lungs and thymus had a dark/red colour. In the other high dose male deaths the lungs, thymus, spleen and thyroid also had a dark/red colour. Histopathology showed moderate to marked myocarditis, centrilobular hepatocyic necrosis with instances of acute inflammation and moderate to marked lymphoid depletion of the thymus were described in both males dosed at 180 mg/kg bw/day and were seen as the possible cause of death. The most important changes observed in the treated female were pulmonary congeastion, lymphocytolysis of the mesenteric lymph nodes and thymus and lymphoid depletion of the spleen. Accidental damage was considered the cause of moderate haemorrhage in the lungs and thymus in one of the two males.

Effect levels

Dose descriptor:

NOAEL

Effect level:

46 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

gross pathology

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In a subchronic toxicity study a NOAEL of 46 mg/kg bw/day was determined following treatment by oral gavage in rats.

Executive summary:

The oral toxicity of Basic Brown 17, when given by daily administration to rats, has been investigated over a period of 13 consecutive weeks and recovery from any potential treatment-related effects over a period of 4 consecutive weeks. Three groups, each of 10 male and 10 female Han Wistar rats, received the test item by gavage at dosages of 60, 120 or 180 mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (distilled water) and acted as a control. Five additional animals for each sex were included in the high and control groups for recovery assessment. The above mentioned dose levels were selected based on information from a preliminary study (RTC Enquiry No.: 30980EXT), in which three groups, each of 5 male and 5 female rats, received the test item by gavage at dosages of 75, 150 or 300 mg/kg/day for 28 consecutive days. A fourth similarly constituted group received the vehicle alone (distilled water) and acted as a control. Mortality : Two males of the control group and 2 males and 1 female of the high dose group were found dead during the treatment period. Pre and post dose observations and weekly clinical signs : At the daily pre- and post-dose observations no signs were detected. At the weekly clinical signs and functional observation tests no significant differences between control and the test item treated animals were seen. Sensory reaction to stimuli and motor activity : There were no changes in motor activity in animals receiving Basic Brown 17 in comparison with controls. Tail pinch response was slightly increased in high dose animals compared with controls at the end of the treatment period but not at the end of recovery. Body weight and food consumption : Body weight and food consumption were unaffected by the treatment. Ophthalmoscopy : No lesions were observed at the end of the treatment. Haematology : No changes of toxicological significance were observed during the study. Clinical chemistry : Two mid-dose females and 3 females and 2 males of the high dose group showed moderate to marked alteration in aspartate aminotransferase. Since alanine aminotransferase was increased only in one high dose female, liver injury could be excluded. No other marked changes were observed. Urinalysis : A number of high dose animals showed bilirubinuria. However, since no hyperbilirubinaemia was observed in the same animals, this test result could be due to the presence of the test item in the urine and/or its metabolites. Terminal body weight and organ weights : There were no differences between control and Basic Brown 17 treated animals in terminal body weight both at the end of treatment and recovery periods. A statistically significant decrease in absolute and relative thyroid weights was seen in high dose females at the end of the recovery period, in comparison with controls. In the same animals the relative liver weight was statistically significantly increased. Macroscopic observations : Unscheduled deaths. One female and 2 males of the high dose group and 2 control males were found dead during the treatment period. The most relevant findings, noted during post mortem examination in both control males and in the treated female, were incomplete collapse and dark/red colour in the lungs, sometimes associated with red staining of the muzzle and/or dark/red colour in the thymus as well as in a number of other tissues. Dark coloration was observed in several tissues/organs from both treated males, one of them also showing firm consistency of the heart, irregular surface and swelling of the liver and oedematous consistency of the prostate. Final sacrifice. Treated animals, mainly those receiving the test item at the high dose level and females appearing more affected than males, showed dark coloration in the brain, heart, kidneys, ovaries, skeletal muscle, spleen and thyroid. Males and females of the mid-dose group also showed dark coloration of the spleen and thyroid. In addition, females showed dark coloration in the heart and skeletal muscle. Recovery sacrifice. Dark coloration was still present after 4 weeks of recovery in the brain, heart, skeletal muscle, spleen and thyroid of treated males and females, when compared with their controls. Microscopic observations : Unscheduled deaths. Moderate to marked myocarditis, centrilobular hepatocytic necrosis with instances of acute inflammation and moderate to marked lymphoid depletion of the thymus were described in the high dose males which were found dead. These changes were seen as the possible cause of death. The most important changes, observed in the treated female which was found dead, were pulmonary congestion, lymphocytolysis of the mesenteric lymph nodes and thymus and lymphoid depletion of the spleen. Moderate haemorrhage of the lungs and thymus, which was considered possibly indicative of a mis-dosing, was described in 1 of the 2 control males. Final sacrifice. Yellow/brown pigmentation was the most relevant treatment-related change observed in the heart, kidneys, liver, spleen, thyroids, Peyer’s patches (ileum) and skeletal muscle of animals, both sexes, mid- and high dose groups, when compared with controls. Yellow/brown pigmented macrophages were also seen in the lungs of mid- and high dose females. In treated males this change was comparable to controls. In addition to the above-mentioned tissues/organs, high dose males and females showed yellow/brown pigmentation in the adrenals, ovaries, uterus, mesenteric/cervical lymph nodes and thymus. Presence or increased incidence of extramedullary haemopoiesis was described in the spleen of animals in all treated groups. Recovery sacrifice. Yellow/brown pigmentation was still detected in the adrenals, heart, kidneys, liver, ovaries, spleen, thyroid, skeletal muscle and mesenteric/cervical lymph nodes from most high dose animals which underwent recovery for 4 weeks. Conclusion : On the basis of these results, the low dose level of 60 mg/kg bw/day (46 mg/kg bw/day active) was considered to be the NOAEL.