A mixture of: N,N-diethylpropane-1,3-diamine 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2,2-iminodiethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate; 2-methylaminoethanol 6-methyl-2-(4-(2,4,6-triaminopyrimidin-5-ylazo)phenyl)benzothiazole-7-sulfonate

Endpoint summary

Description of key information

For the oral route, a 28 day subacute toxicity study (RCC 255571) accompanied by a 5 day range finder (RCC 205582) are available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

In an oral range finding study which followed no guideline, the test article was administered to rats by repeated oral gavage, for a period of 5 days (RCC205582).The aim of this study was to provide a basis to propose dose levels for a 28-day oral toxicity study.

The study comprised three groups, each containing three male and three female rats, and the dose levels tested were 0, 200 and 1000 mg/kg bw/day.

Neither mortality nor clinical signs were observed. No differences which could be directly related to test article treatment were observed for body weight and food consumption. Ophthalmoscopic examination revealed no treatment-related findings . No macroscopic organ changes were observed. The absolute kidney weights and liver weight ratios of the male rats of the high-dose group were significantly increased when compared with the control and low dose group.

The absolute and relative liver and kidney weights of the female rats of the high-dose group were significantly increased when compared to the results of the control and the low dose groups. These findings might be an effect of the higher initial body weight of these animals. Based on the results of the present 5 day study, the following dose levels appeared to be appropriate for a subacute 28-day oral (gavage) study: 0, 50, 200 and 1000 mg/kg body weight.

In the subacute 28-day toxicity study conducted according to the OECD TG 407, the test article was administered daily by gavage to Wistar rats of both sexes (RCC 205571) at the dose levels mentioned above; 50 mg/kg bw/d was designated as low dose, 200 mg/kg bw/d was designated as mid dose, and 1000 mg/kg bw/d was designated as high dose.

The rats were assigned to toxicity and recovery testing per group as follows:

Group 1 (control): 10 animals/sex were used and treated during the 28 days testing period; 5 animals /sex were kept after ending of the treatment period for another 14 day recovery period.

Group 2 (low dose, 50 mg/kg bw/d): 5 animals/sex were used and treated during the 28 days testing period.

Group 3 (mid dose, 200 mg/kg bw/d): 5 animals/sex were used and treated during the 28 days testing period.

Group 4 (high dose, 1000 mg/kg bw/d): 10 animals/sex were used and treated during the 28 days testing period; 5 animals /sex were kept after ending of the treatment period for another 14 day recovery period.

No mortality occurred; all animals of the high dose group showed orange discoloured faeces between days 5 and 28 of the treatment period. The animals of the recovery group showed the same discoloration between days 1 and 3 of the recovery period (test days 29-31) but had recovered from day 4 of recovery (day 32 of test).

No statistically significant differences in body weight and body weight gain were observed during treatment and during recovery. No statistically significant differences which could be related to test article treatment were observed throughout the entire test and recovery period when the food consumption of the control animals was compared to those of the animals of the test article-treated groups. Ophthalmoscopic examinations revealed no treatment-related effects in the eyes of the treated animals.

The assessment of haematological data indicated a slightly decreased erythrocyte count, haemoglobin concentration and hematocrit value for both sexes of the high dose group at 4 weeks of treatment. At the end of the recovery period the findings were found to be reversible but still slightly below that of the controls. The findings reflect a slight anaemic condition and are therefore considered treatment-related.

The assessment of clinical chemical parameters revealed following effects in the high dose group at the end of the 4 week treatment: decreased glucose and increased urea level for males, increased triglyceride level for females, decreased phospholipids level for males, increased aspartate aminotransferase activity for both sexes, increased calcium level for females, increased sodium and chloride level for both sexes, increased potassium level for males, increased albumin level for both sexes and increased total protein level for females. The changes however were only slight when compared to controls and completely reversible after 14-day recovery; thus, they were not considered significant in toxicological terms, but a reflection of stress induced metabolic adaptation due to the treatment.

Urinalysis data indicated a slightly decreased overnight urinary output (volume/18 hrs) for males and slight proteinuria for females of the high dose group after 4 weeks of treatment; the findings were reversible at the end of the recovery period. However, it remains unclear whether these findings were treatment-related.

Statistically significant increased absolute and/or relative kidney weights were observed in male and female rats of the mid dose group (females only) and the high dose group 4 after the 4 week treatment and also after recovery. In addition the high dose females showed statistically significant increased absolute and/or relative liver weights upon sacrifice at test ending (4 weeks) and after recovery. These findings were considered a metabolic adaptation of the organs and therefore not significant in toxicological terms.

Since treatment-related effects were seen in the high dose group treated with 1000 mg/kg bw/day, the LOAEL was set at 1000 mg/kg bw/day. The NOAEL was set at 200 mg/kg bw/day for both, male and female Wistar rats treated orally by gavage with the test article for 28 days.

Justification for classification or non-classification

Based on the results of a validated 28 day subacute oral toxicity study, the LOAEL for the test article was set at 1000 mg/kg bw/day. Thus, there is no need for classification of the test article according to the EU Directive 67/548/EEC and to the CLP Regulation EC/1272/2008.