A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate

Endpoint summary

Description of key information

NOEL (oral, 28 d) = 50 mg/kg/day

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

haematopoietic

Organ:

spleen

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

The repeated dose toxicity of the substance was evaluated in a subacute 28-day toxicity study, according to the OECD Guideline 407 (1981) and method B.7 of the EEC-Directive 92/69 EEC.

A 5-day range finding study was performed (with 3 rats/sex/group at dose levels of 50, 200 and 1000 mg/kg/day) to provide a basis for selection of dose levels for a study of longer duration.

With the exception of dark appearance of the feces, no differences of biological significance were apparent between animals of the 3 treatment groups.

Based on these observations, a high treatment level of 1000 mg/kg/day was selected for a study of 28 days duration.

In the main study, the test item was administered dally by gavage to SPF-bred Wistar rats. The study comprised of four groups. The number of rats assigned to toxicity testing per group as well as the dose levels administered were as follows:

group	Dose level mg/kg	males	females
1	0	1-5	21-25
2	50	6-10	26-30
3	200	11-15	31-35
4	1000	16-20	36-40

The following findins were observed.

At 50 mg/kg/day: No treatment-related changes detected.

At 200 mg/kg/day:

1)  Dark appearance of the faeces was noted in males and females.

2)  Microscopically observed accumulatlon of iron pigments were noted in the spleen of males and females.

At 1000 mg/kg/day:

1)  Dark appearance of the faeces was noted in males and females

2)  Increased total white blood cell counts were noted in males and females,

3)  Increased spleen weights and relative spleen weights were noted in males and females and increased relative kidney weights were noted in males only.

3)  Microscopically observed accumulatlon of iron pigments and increased number of haemopoletic foci were noted in the spleen of males and females.

From the results presented in this report a definitive no observed effect level (NOEL) of 50 mg/kg/day was established.

Justification for classification or non-classification

According to the CLP Regulation (EC) no. 1272/2008, classification in Category 1 for repeated dose toxicity applies to substances that have produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure. Substances are classified in Category 1 for target organ toxicity (repeat exposure) on the basis of:

— reliable and good quality evidence from human cases or epidemiological studies; or

— observations from appropriate studies in experimental animals in which significant and/or severe toxic effects, of relevance to human health, were produced at generally low exposure concentrations.

Classification in Category 2 applies to substances that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to be harmful to human health following repeated exposure. Substances are classified in Category 2 for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant toxic effects, of relevance to human health, were produced at generally moderate exposure concentrations. If classification is based on results obtained from studies conducted in experimental animals, the dose/concentration guidance values that classify refer to effects seen in a standard 90-day toxicity study conducted in rats. These guidance values are presented in the CLP Regulation (EC) no. 1272/2008) in Annex 1: 3.9.2.9.6., Table 3.9.2 for classification in Category 1 and in Annex 3.9.2.9.7., Table 3.9.3 for classification in Category 2. For a 28-day study the guidance values is increased by a factor of three.

As the only effects observed at 200 mg/kg/day in the Repeated Dose 28-Day Oral Toxicity Study were dark faeces and iron pigments in the spleen, and as these may not be considered adverse effects of systemic toxic relevance, 200 mg/kg/day can be extrapolated to be the LOEL value of the substance. Therefore, the NOAEL is expected to be greater than this value, and at 300 mg/kg/day the eventual effects are expected to be more comparable to those at 200 than 1000 mg/kg/day. Therefore, no classification for repeated dose toxicity is warranted under the CLP Regulation (EC) no. 1272/2008.