alkoxy substituted aryl diazo heterocyclic aryl nitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: Disperse Yellow DYLA 1306

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION
ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: no in vivo studies are available for the substance to adequately address this endpoint. There is a negative in-vitro gene mutation assay (HPRT) in mammalian cells (V79) available, showing that no mutagenic effects were seen in mammalian cells.

- Available non-GLP studies: no in vivo studies are available with the substance to adequately address this endpoint.

- Historical human/control data: There is no historical human data available for this substance on ‘genetic toxicity in vivo’. Hence no evidence of human adverse effect.

- (Q)SAR: (Q)SARs are not considered adequate to address this endpoint. As nitro-compounds usually are Ames positive due to bacterial reduction of the nitro-group, QSAR prediction would have the same outcome for this endpoint and there is not sufficient information on these structures from in-vivo studies.

- In vitro methods: Indication for a bacteria-specific effect due to bacterial nitro-reductase can be drawn from running a modified Ames test with nitro-reductase negative strains. These strains are available at some testing laboratories for Salmonella strains TA98 and TA100. However, although these strains are named nitro-reductase negative, they still have a low amount of nitro-reductase and thus most of the time, only a decrease in the positive effect and no clear negative result is seen. No such study is available with the substance.
Umbuzeiro et al., 2005 investigated this effect in a different azo dye carrying the nitro-group, showing in a modified Ames test that the substance is positive in nitro-reductase and O acetyltransferase positive Salmonella typhimurium strains, but negative with nitro-reductase and O acetyltransferase negative strains (Umbuzeiro et al., 2005). In this publication, Disperse Blue 291 (EC 260-255-0) was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitro-reductase and O acetyltransferase (i.e., TA98DNP6, YG1024, and YG1041) as well as standard strains TA1535, TA1537, TA1538, TA98 and TA100 and strains which provide more information on the base-pair substitution (TA7001 to 7006). According to this publication, Disperse Blue 291 showed direct-acting mutagenic activity with all strains of Salmonella typhimurium tested, except for TA1535. According to the classification of Claxton et al. (1991), the potency of this substance can be considered moderate (10-100 revertants/µg). In the absence of S9, the nitro-reduction is strongly related to the mutagenic activity, because the mutagenicity of Disperse Blue 291 was very low when tested with the strains lacking nitro-reductase activity (TA98NR) and was increased with the nitro-reductase overproducing strains, (YG1021 and YG1041). The same mutagenic pattern was observed for the O acetyltransferase deficient and overproducing strains (TA98DNP6, YG1024 and YG1041) revealing also the importance of the O acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitro-reductase and O acetyltransferase overproducing strain (YG1041), it is likely that the product of the nitro-reductase is a substrate for the O acetyltransferase. This study is hence clearly showing that the increase in mutation frequencies is a bacteria-specific effect.

- Weight of evidence: For the substance, a weakly positive Ames test conducted with Salmonella strains TA98, TA100, TA1535 and TA1537 and E. coli WP2 uvrA and a negative mutagenicity assay in mammalian cells is available. In the Ames test, a weak positive effect was seen in TA100 in the presence of the metabolising system at either the highest (first assay) or the two highest (second assay) concentrations in the presence of precipitation.
Weight of evidence is given that this positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells. The nitro-reductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitro-reductase proteins play a central role in the activation of nitrocompounds [de Oliveira et al. 2010]. Type I nitro-reductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitro-reductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group.

The fact that this effect was only observed with metabolic activation and only in Samonella typhimurium TA100, is due to the fact that the metabolic activation of the aromatic amine group has been shown to be primarily prevented by steric hindrance [Klein et al 2000; Kazius et al 2005]. Hence, the nitroreductase could only activate the structure after the protective group has been removed by enzymes of the metabolising system. However, this is only the case in one Salmonella strain with plasmid pKM101, whose mucAB gene products enhance SOS mutagenesis; which makes strain TA100 more sensitive for mutagen detection [Prival et Zeiger 1998].

- Grouping and read-across: no structural analogues were identified with data on in-vivo mutagenicity

- Substance-tailored exposure driven testing: not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The ECHA guidance R.7a states that following a positive result in an in vitro test, “adequately conducted somatic cell in vivo testing is required to ascertain if this potential can be expressed in vivo.”
Although the weight of evidence assessment clearly indicates the involvement of bacterial enzymes in the positivity of the test, the available data do not prove that the observed minor effect is exclusively due to this mechanism.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design: According to OECD 489 in liver, glandular stomach and duodenum of one sex only, as no toxicologically relevant differences were observed between males and females.
No germ cells will be collected, as the assay is not validated for germ cells, the TG states "this guideline is not considered appropriate to measure DNA strand breaks in mature germ cells. Since high and variable background levels in DNA damage were reported in a literature review on the use of the comet assay for germ cell genotoxicity".

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

GLP compliance:

yes

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Test item: Disperse Yellow DYLA 1306

Test animals

Species:

rat

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Duration of treatment / exposure:

2 treatments (24 h and 4 h prior to preparation)

Frequency of treatment:

2 treatments

Post exposure period:

24 h and 4 h

No. of animals per sex per dose:

6 (one sex, as there is no gender difference)

Control animals:

yes, concurrent vehicle

Positive control(s):

yes

Examinations

Tissues and cell types examined:

A) Analysis of hepatocytes
Isolation of hepatocytes by mincing
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)
B) Analysis of cells from the stomach
Isolation of cells from the stomach by scraping
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)
C) Analysis of cells from small intestine
Isolation of cell nuclei from the small intestine by mincing
Determination of dead cells in 500 cells per slide (1500 per animal)
Performance of the alkaline comet assay
Analysis of 50 cells per slide (150 cells per animal)