A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)

Endpoint summary

Description of key information

Oral subacute toxicity study (28 -day) with rat: NOEL = 200 mg/kg bw/day; NOAEL = 1000 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study was performed according to OECD TG 407 and GLP.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subacute toxicity via oral route

A study was run according to OECD guideline 407. The substance was administered daily by gavage to rats. The study was run with four dose groups, 0, 50, 200, and 1000 mg/kg/day.

At 50 and 200 mg/kg/day no treatment related findings were observed.

Following observations were made at 1000 mg/kg/day:

1) dark faeces and regurgitation of test substance.

2) decreased body weight gain in females.

3) decreased food consumption by females.

4) increased liver weight in females only.

Animals receiving 1000 mg/kg showed increased liver weights (although not statistically significant in males). However, as there were no changes in serum liver enzyme levels or pathological evidence to corroborate this, the toxicological significance of this change has to be considered doubtful. The decreased food consumption by females receiving 1000 mg/kg/day noted even after adjustment for body weight suggests a loss of appetite among these females rather than any impairment of metabolic function.

Given these slight, but unequivocal, indices of biological effect among animals treated at 1000 mg/kg/day, a no observed effect level (NOEL) was identified at 200 mg/kg/day, and the NOAEL at 1000 mg/kg bw.

Repeated dose toxicity via inhalation route

The inhalation route is appropriate if exposure of humans via inhalation is the most relevant route of human exposure taking into account the vapour pressure of the substance and/or the possibility of exposure to aerosols, particles or droplets of an inhalable size.

The very low vapour pressure applies to test item, as the study on vapour pressure determination was not run based on the high melting point > 300 °C. The particle size distribution shows D90 76.7 µm, D50 37 µm and less than 5 % < 10 µm, thus most particles belong to the inhalable fraction (10-100 µm). If inhaled, particles remain in the high part of the respiratory tract, from which they could be cleared out or enter the gastrointestinal tract. Possible toxic effects may be assessed in a toxicity study by oral route. Furthermore, significant human exposure to the substance via inhalable particles or droplets through the inhalation route is deemed to be unlikely due to the use of the substance in form of aqueous solutions as a dye.

Therefore, a study on repeated dose toxicity via the inhalation route was considered as not appropriate.

Repeated dose toxicity via dermal route

The dermal route is appropriate if the physico-chemical properties suggest potential for a significant rate of absorption through the skin.

Looking at the data set and the structure of the molecule, it can be postulated that this material has very little bioavailability by the dermal route. No systemic effects have been seen in an acute toxicity study up to a dose level of 2000 mg/kg bw; positive response was seen in 1 out of the 2 skin sensitisation studies, indicating bioavailability of the substance. However, as skin sensitisation is a non threshold effect, such evidence is not in contrast with the assumption of low absorption and bioavailability of the substance. Furthermore, local dermal effects caused by the substance have adequately been characterised via in vivo studies on skin sensitisation, skin and eye irritation and acute dermal toxicity. Overall, a study on repeatd dose toxicity via dermal route was considered as not appropriate.

Subchronic toxicity via oral route

The substance is poorly absorbable and has low toxicity, i.e. LD50 > 5000 mg/kg and NOAEL of 1000 mg/kg bw/day from 28-day repeated dose toxicity study and from developmental toxicity study. According to literature data and QSAR predictions, toxicity of azo-dyes is related to the potential release of aromatic amines upon cleavage of the diazo bonds in metabolic processes. There is no evidence of significant metabolism of the substance. Indeed, the release of the main metabolic products, such as those expected based on molecular structure as well as on QSAR predictions, would imply a high toxicity upon both acute and prolonged exposure, based on available data. None of these signs is seen upon exposure to target substance in various studies: acute toxicity study, 28-day repeated dose toxicity study, developmental toxicity study. Consequently, the formation of such metabolites is believed as not relevant under in vivo conditions. Physico-chemical features of the substance limit the absorption rate and thus the possibility of metabolism, in particular this is due to a high molecular weight (ca. 1090 g/mol), a high solubility in water (1:1 miscibile in water), a low partition coefficient (logPow < 0).  In conclusion, a 90-day repeated dose toxicity study is not expected to modify the toxicological profile of the substance and further testing on vertebrate animals for this endpoint is proposed to be omitted.  

Justification for classification or non-classification

Based on the experimental evidences in the assessment of the toxicity potential after repeated oral exposure, the test item does not need to be classified according to the CLP Regulation (EC 1272/2008).