Disodium; 4-Amino-3-{4-[4-(2,4-diamino-phenylazo)-benzenesulfonylamino]-phenylazo}-5-hydroxy-6-phenylazo-naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

The value of NOAEL for repeated dose toxicity is 150 mg/kg bw/day based on minor modulations observed in both genders, such as slight changes in the pH of urine, spleen enlargement and colour by the test substance, with no other overt evidence of tissue damage, dysfunction, or increased tissue weight.

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

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Two tests are available on similar substance 1 and considered reliable to cover also the endpoint for Acid Black 234.
In the first study (Consortium, 2011) Similar substance 1 was tested for 42 day repeated oral administration to wistar rats by gavage at the dose levels of 50, 150 and 450mg/kg bw/day. At these doses neither mortality nor overt signs of toxicity or other modulation in functional observation were observed in both genders of each group. In particular the only effect observed up to the dose of 150mg/kg bw/dayin both genders was an increased pigmentation (dark-grey colour) along with slight enlargement of the spleen. However, nor increase in spleen weight neither significant modulations have been observed in both haematological and biochemical parameters of both genders. For these reasons, though the modulation observed in spleen is likely to be due to repeated exposure to the test compound no other detectable adverse alterations of morphology, functional capacity, growth, development, or life span were observed. Thus, the value of NOAEL (No Observed Adverse Effect Level) was established as150 mg/kg bw/day in both genders. On the contrary, the dose of 450 mg/kg bw/day exerted significant modulations both in haematological and biochemical parameters. as well as increased occurrence of dark coloured kidneys, spleen, liver, lymph nodes and ovaries. Moreover at the highest dose level significant alteration of absolute spleen and brain weight was also observed.
Another study was available on similar substance 1 (Stal, 1996) following EU method B7 with oral administration. The NOAEL was found to be 150 mg/kg bw/day and NOEL equal to 25 mg/kg bw/day.
The two tests show the same level of toxicity (as NOAEL) for repeated dose toxicity testing, equal to 150 mg/kg bw/day. This value was then used for CSA.
A Dose Range Finding, following OECD 422 has been performed in the target substance, Acid Black 234, in order to confirm the similarity of the systemic effects with the analogue substance Acid Black 210.
The tests showed that at the highest dose tested (1000 mg/kg/d) there was an unplanned death of all females and males during the first week of study (cause unknown). Anyway, at the intermediate dose 450 and 150 mg/kg/d, minor effects are seen in comparison with Acid Black 210.
Minor statistically significant differences were showed in haematological examination. In males, at the dose levels 150 and 450 mg/kg/day was observed decrease of total erythrocyte count, which was associated with the decline decreased of haemoglobin, haematocrit and increased of mean corpuscular volume. In females were detected decrease of total erythrocyte count and mean corpuscular volume at the dose level 450 mg/kg/day .
Macroscopical structure of organs of males, females and foetuses – in all males at the dose level 450 mg/kg/day were found dark enlarged spleen. In all females at the same dose level were found dark spleen. No macroscopic changes were found during the pathological examination of foetuses.
Subsequent histopathological examination of spleen in males at the dose levels 150 and 450 mg/kg/day and in females at the dose level 450 mg/kg/day revealed siderosis, extramedullary hemopoiesis and venostasis. Testicles in all dose levels did not show any pathological findings.
Read across evaluation is attached in section 13.

Justification for classification or non-classification

Regulation "EC 1272/2008 on classification, labelling and packaging of substances and mixtures", regarding classification criteria for substances (Annex I, table 3.9.1) states that "Substances are classified in category 2 for target organ toxicity (repeated 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".

Moreover, at paragraph 3.9.2.9.2 it reads:" In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified, dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. Repeated-dose studies conducted in experimental animals are designed to produce toxicity at the highest dose used in order to optimise the test objective and so most studies will reveal some toxic effect at least at this highest dose. What is therefore to be decided is not only what effects have been produced, but also at what dose/concentration they were produced and how relevant is that for humans". These "guidance values" are provided in table 3.9.3, and refers to effects observed in a standard 90 day repeated dose study in which classification is not applicable when "significant toxic effects" are detected over a dose of 100 mg/kg/day. Then by using dose/exposure extrapolation similar to Haber's rule for inhalation which states that "the effective dose is directly proportional to the exposure concentration and the duration of exposure" we might put forward the hypothesis that classification is not applicable in a 42 day repeated toxicity study if significant toxic effects are observed over a dose of about 150-200 mg/kg/day.

The repeated oral toxicity test (42 day) of the present study did not exert significant toxic effects in morphology, growth, central nervous system depression of animals up to the dose of 450 mg/kg/day.

Moreover, neither death nor moribund conditions were reported up to this dose for all animals of both genders. These observations might indicate a low bioaccumulation of the compound or its metabolites.

This suggests that though significant changes were on the contrary observed in haematological and biochemical parameters at 450 mg/kg/day, the detoxification process was not overwhelmed even after repeated oral exposure to doses not relevant for predicting human risk.

Moreover, even by using the precautionary value of NOAEL of this study (150 mg/kg/day) for the classification of the substance, we should stress here that effects observed were considered not to support classification. In fact after exposure to 150 mg/kg/day only minor modulations have been observed in both genders, such as slight changes in the PH of urinalysis, spleen enlargement and coloured by the testing substance, with no other overt evidence of tissue damage, dysfunction, or significant increased tissue weight.

In conclusion, since no data on human epidemiology or incidental exposure to acid black 234 are available, and giving the nature and severity of effects of prolonged exposure to doses which are significantly higher than possible human exposure (See chapter 9 and 10 of CSR), we do not see a consistent and identifiable toxic effect which demonstrates need for the classification.

No classification for repeated dose toxicity is warranted under Regulation 1272/2008