A mixture of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-); tert-alkyl(C12-C14)ammonium ((1-(4(or 5)-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-pentylphenylazo)-2-naphtholato))chromate(1-)

Endpoint summary

Description of key information

The NOAEL for the test substance was determined at 50 mg/kg bw/d in a repeated dose (28-d) toxicity study in rats including a 14-day recovery period (OECD 407, GLP). At 200 mg/kg bw, males had a slightly reduced reticulocyte count. At 1000 mg/kg bw, males and females had a reduced reticulocyte count and a few other changes in heamatology. Two of ten females focal cortical hypertrophy in the adrenal gland. All findings were reversible during the 2-week recovery period.

Key value for chemical safety assessment

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

Additional information

In a repeated dose (28 -day) oral toxicity study according to OECD TG 407 and GLP performed in 1988, the test substance was administered daily by gavage to Sprague-Dawley rats at dose levels of 0, 50, 200 or 1000 mg/kg bw/d for a period of 4 weeks. Additional five animals per dose group were kept for a 14 day treatment-free recovery period.

Repeated oral intake of the test substance at 1000 mg/kg/ bw/day for some animals resulted in purple or blue skin/fur as well as in bluish discolouration of the pancreas and testes. However, in the absence of any microscopic correlates and given that the test article is a dye stuff, these discolourations were concluded to be due to passive staining and not a toxicological response. The bluish discoloration of the pancreas and testes was observed in three of five males after 28 days. At the end of the recovery period, one male showed bluish discoloration in testes. For females, one of five and none of five animals showed bluish discoloration of the pancreas at the end of the treatment period and after recover, respectively.

Treatment related adverse indings involve haematology and histopathology of the adrenal cortex and were reversible within the 14 -day recovery period. The nature of findings does not allow a conclusion on a target organ or mode of action. The most sensitive parameter was the dose-dependent reduction in reticulocyte count which was observed at 200 mg/kg bw in males. All other findings occured at 1000 mg/kg bw only.

In males, an increased number of large unstained cells was observed.

An increased blood serum bilirubin level in males as well as females was not confirmed by histopathological examination or urinary clearance. The cortical hypertrophy in the adrenal glands was limited to two females. However, a dose related response was not evident. The slight increase of white blood cells in the urine was limited to males of the high dose group only and, in the absence of any other corroborative change, considered to be of equivocal toxicological significance. Based on the results in this study a NOAEL of 50 mg/kg bw/d was determined.

In contrast to the above described 28 -day study, mortality/morbidity was observed in parental animals of the reproductive toxicity screening study at dose levels of 1000 and 500 mg/kg bw. Higher toxicity correlated with an earlier onset and a more intense black disocoloration of animals. As the substance is a black dye, the coloration is considered to be a passive effect of the colorant. In order to examine this unexpected toxicity and relate it to a possible excess of free primene, a washed sample was given to male rats. The washing step did not change the fact that animals were stained completely black after three days of treatment with 1000 mg/kg bw. Upon cessation of dosing, the black coloration disappeared.

No reason for the differences in toxicity profiles could be elucidated from clinico-chemical and haematological investigations performed with blood taken after 14 days of dosing.

	Subacute oral tox 1988OECD 407(no mortality)	Reproductive Screen 2013OECD 421(mortality)	follow-up test study in three rats (purified, 1000 mg/kg bw)(moritality)
External body coloration	purple appearance (grade 1 - minimal) over the whole body of all females and males treated at 1000 mg/kg/day	black at 1000, 200, 50 mg/kg bw (dose-dependent, including eyes)	yes, black (including eyes)
onset of discoloration	day 12	starting day 2, whole body black by day 5	starting day 1 (grey, skin and eyes), completely black on day 3 in all animals
reversibility of discoloration	yes, duration 7 days, going via a blue colour (grade 1 -minimal)		yes, via grey shading
Complete Internal body coloration	none after 28 days	At 1000 mg/kg bw after 7 days	yes, completely
		At 200 and 50 mg/kg bw after 28/54 days
Organ discoloration	Several rats from the high dose group showed bluish discolouration of the pancreas and testes.	yes	yes
Blood parameters	reduced reticulocytes after 28 days, reversible	No effects after 2 weeks	not examined
Clincical Chemistry	bilirubin increased at 1000 mg/kg bw after 28 days, reversible	No effects after 2 weeks	not examined
Histopathology	severe degree of focal cortical hypertrophy in the adrenal glands of two high dose female rats	No effects after 7 days (1000 mg/kg bw), no effects after 28/54 days (few organs only)	not examined

Justification for classification or non-classification

The findings of the subacute oral toxicity study at 50, 200 or 1000 mg/kg bw are neither serious nor irreversible. No target organ of systemic toxicity could be identified.

Therefore, the test substance is not subject to classification and labelling for repeated dose or specific target organ toxicity according to Directive 67/548/EEC and Regulation 1272/2008/EC.