Reaction mass of bis[3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-) and bis[5-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-)

Endpoint summary

Description of key information

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of Basic Red 46 Tetrachlorozincate. The data indicate that there is little or no dermal absorption. No signs of a significant systemic toxicity associated with absorption through skin have been observed. Based on exposure model from AG Textilien des Bundesinstituts fur Risikobewertung (BfR), the dermal penetration rate for dyes through the skin was found to be less than 2%. However, a factor of 25 (4% dermal absorption) was taken into consideration as worst case for the oral to dermal route to route extrapolation. Based on physico-chemical data and the results of oral toxicity studies, Basic Red 46 has a relatively good oral bioavailability.

The substance is considered to have low volatility as evident from the melting point at ca. 207°C, so the potential for the generation of inhalable forms is low. The molecular weight of the single chromophore is 321.4 g/mol and the chromophore is positive charged. This together with the high water solubility (34.3 g/L) and low partition coefficient value of ca. -1.3, indicate the substance is not able to cross the mucous layer of the respiratory tract .Due to the high water solubility, vapours if generated/inhaled, will be trapped in the mucus of the respiratory tract, thereby further limiting the absorption. Hence, the main route of exposure of the substance, if inhaled, will be due to swallowing of particles deposited in the nose/mouth. Therefore, the bioavailability for inhalation is considered the same as for oral intake.

Bioaccumulation of Basic Red 46 can most probably be excluded due to the available data. Based on the results of genotoxicity assays, a metabolisation towards genotoxic metabolites can also be excluded for mammalian species.

The substance is therefore not considered to be of concern for ADME related effects.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

4

Absorption rate - inhalation (%):

100

Additional information

Introduction

Toxicokinetic parameters such as uptake, distribution, metabolism and excretion form the essential toxicological profile of a substance. An approximate indication of the toxicokinetic pattern can be gained from the physico-chemical properties taking into account the molecular weight, the number of atoms (hydrogen bond donors and acceptors), the solubility in solvents, log K_OW, etc. and the results of basic toxicity testing of the test article. The assessment of the toxicokinetic properties of Basic Red 46 Tetrachlorozincate given below is based on the results obtained for, the following toxicological endpoints with Basic Red 46 with various counter-ions (ZnCl₄, Br, methyl sulfate):

• Acute oral toxicity in rats
• Acute dermal toxicity in rats
• In vivo skin irritation in rabbits
• In vivo eye irritation in rabbits
• Skin sensitization in guinea pigs
• Bacterial reverse mutation test
• In vitro mutagenicity assay in mammalian cells (Basic Red 46 Br)
• In vitro chromosome aberration assay in mammalian cells (Basic Red 46 Br)
• Subacute oral toxicity in rats (Basic Red 46 methyl sulfate)
• Reproductive screening study in rats (Basic Red 46 methyl sulfate)

Allstudieswere carried out according to the principles of Good Laboratory Practice and/or met the requirements of the OECD and EU-Guideline for the Testing of Chemicals.

Substance identity and physico-chemical properties

Name:                             Basic Red 46 Tetrachlorozincate

EC number:                     916-918-8

EC name:                        Reaction mass of bis[3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-) and bis[5-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-)

Physical state:                  black solid

Empirical formula:           C₃₆H₄₂N₁₂Cl₄Zn

Molecular weight:            ≤ 850 g/mol                                           (<500 daltons=good absorption)

                                       321.4 g/mol (chromophore)

                                       425 g/mol for C₁₈H₂₁N₆.Cl₂Zn_0.5

Water solubility:              34.3g/L                                                (= soluble in water)

Partition coefficient:         log Kow -1.3                                         (>-0.4 or <5.6 = good absorption)

Atom count (natoms):      24                                                         (<70 = good bioavailability)

H-bond acceptor (nON):   6                                                           (<10 = good bioavailability)

H-bond donor (nOHNH):  0                                                           (<5 = good bioavailability)

Toxicological profile

Acute oral toxicity of Basic Red 46 Tetrachlorozincate was evaluated in male and female rats at dose levels of 562, 825, and 1210 mg/kg bw. Adverse effects observed consisted in bad general condition, dyspnoea, sedation, ataxia, salivation and red stained urine. The oral median lethal dose was calculated to 934 mg/kg bw based on test material which equals 560.4 mg/kg bw based on 100% active ingredient. No mortality or adverse effects were seen in the dermal toxicity study at the limit dose of 2000 mg/kg body weight. Furthermore, no systemic toxicity was observed during testing for skin sensitising and skin or eye irritating properties of Basic Red 46 Tetrachlorozincate in guinea pigs and rabbits, respectively. Basic Red 46 was not irritating to skin, but caused irreversible damage to eyes and proved not to be skin sensitising in guinea pigs.

The reproductive and repeated dose toxicity of the test substance (Basic Red 46 methyl sulfate) was determined in male and female rats in a combined repeated dose and reproductive-developmental screening study according to OECD Guideline 422, in compliance with GLP. Rats were administered the test substance orally by gavage at dose levels of 25, 75 and 175 mg/kg body weight/day in the main study. Clinical signs were detected in animals of either sex from all treatment groups. Episodes of increased salivation and noisy respiration were evident throughout the study. In addition, fur stained by the test item (pink/red) and pink stained bedding was observed. Impaired body weight development was observed throughout the study in test substance treated males. Subsequently, a reduction in overall body weight gain was evident in treated males and reductions in food consumption and food conversion efficiency was also evident throughout the treatment period for males treated with 175 mg/kg bw/day. Females treated with 175 mg/kg bw/day also showed reductions in body weight gains during maturation, the final two weeks of gestation and during lactation and food consumption was reduced in these females during lactation. Observations of this nature are often reported when a test item formulation is unpalatable or irritant and can be associated with gastric irritancy rather than attributable to true systemic toxicity. This was supported microscopically in animals of either sex treated with 175 mg/kg bw/day and in males treated with 75 mg/kg bw/day, where hyperplasia and hyperkeratosis of the non-glandular region of the stomach was evident. Based on the above findings, the ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 75 mg/kg bw/day for females and 25 mg/kg bw/day for males. The reduced body weight development and microscopic changes evident in the stomach were considered to be the result of an irritant effect of the test item rather than a true systemic effect and as such was considered adaptive and non-adverse. Further the microscopic changes observed were seen in the non-glandular region of the rodent stomach. The corresponding non-glandular area is not present in the man and hence the adverse effects were considered not significant to the man. Therefore, a ‘No Observed Adverse Effect Level’ (NOAEL) can be established at 175 mg/kg bw/day for animals of either sex.

The test substance was tested in a reverse gene mutation assay in bacteria (equivalent to OECD 471), in strains TA 1535, TA 100, TA 1537, TA 1538 and TA 98 of Salmonella typhimurium. The substance was found to be mutagenic in bacteria tester strains S. typhimurium TA 98 and TA 1538 at cytotoxic concentrations in the presence of an exogenous metabolic activation system. Basic Red 46 was tested for mutagenic effects on V79 Chinese hamster cells in vitro with and without metabolic activation. Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in all treatment series. No relevant increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolism. Basic Red 46 was further tested in a chromosome aberration test in V79 Chinese hamster cells in vitro with and without metabolic activation. No clastogenic effect was observed in the assay with V79 Chinese hamster lung fibroblasts neither in presence nor absence of an exogenous metabolic activation system.

Evaluation and Assessment

The test item is a has a moderately high molecular weight (425 g/mol; 321.4 g/mol for the chromophore). Based on the modified Lipinski rule of five, a relatively good oral absorption is expected. This can be verified by the results of acute and repeated toxicity studies, which have demonstrated that there is good oral absorption of Basic Red 46 upon exposure, as seen by the toxic effects and the red staining of the urine. On the other hand, based on the chemical structure and molecular weight, it is predicted that Basic Red 46 has limited dermal absorption which is confirmed in the acute dermal and local toxicity studies. Basic Red 46 has a low volatility and therefore, no significant uptake of the substance via inhalation is expected.

For any test item that is absorbed following oral ingestion, the high water solubility should facilitate the distribution of the test substance throughout the body in the water compartment of circulatory systems. The chemical structure of the test item, with its lack of significant reactive groups on the molecule suggest that the test item will not bind to proteins within the circulatory system. Accumulation of the test substance in body fat is not expected due to the high water solubility and low octanol: water partition coefficient value. The passage of test item across specialized biological membranes such as the blood/brain, blood/testis or blood/placental barrier will be subject to the same physical limitations (high molecular weight and low octanol: water partition coefficient) as with absorption from the gastro-intestinal tract and may therefore suggest that biological distribution will be inhibited across such biological structures. This suggestion may be supported by the lack of test item stained cellular structures such as brain and testis identified from histopathological assessment of treated animals from the repeated dose toxicity study.

According to the molecular weight, Basic Red 46 is most likely predominantly eliminated via faeces – as it has been shown that substances with molecular weight above 300 g/mol are preferentially excreted via faeces in rats. However, as the acute and repeat-dose studies showed, a fraction of the absorbed test substance and most likely its metabolites are excreted via urine.

Summary

The results of basic toxicity testing give no reason to anticipate unusual characteristics with regards to the toxicokinetics of Basic Red 46 Tetrachlorozincate. The data indicate that there is little or no dermal absorption. No signs of a significant systemic toxicity associated with absorption through skin have been observed.Based on physico-chemical data and the results of oral toxicity studies, Basic Red 46 has a relatively good oral bioavailability.Bioaccumulation of Basic Red 46 can most probably be excluded due to the available data. Based on the results of genotoxicity assays, a metabolisation towards genotoxic metabolites can also be excluded for mammalian species.

The substance is therefore not considered to be of concern for ADME related effects.