Reaction products of 2-amino-4-chlorophenol, 2-amino-4-nitrophenol-6-sulfonic acid with resorcinol, iron complex, sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Gene Mutation study in Bacteria -AMES

The test item induces reverse mutation in Salmonella typhimurium, both in the absence and presence of S9 metabolism, under the reported experimental conditions.

MUTATION IN L5178Y TK+/ − MOUSE LYMPHOMA CELLS (screening test)

The substance induced mutation at the TK locus of L5178Y mouse lymphoma cells cultured in vitro in the absence and presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

In vitro gene mutation study in bacteria (AMES)

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with Phenobarbital and 5,6-Benzoflavone, in Main Assay I and Main Assay III, and liver S9 fraction from uninduced hamsters(reductive metabolic activation system with Prival modification), in Main Assay II.

The test item was used as a solution in sterile water for injection and, as requested by the Sponsor, concentrations were expressed in terms of active ingredient.

Toxicity test

5000, 1580, 500, 158 and 50.0 µg/plate, standard metabolic activation system.

No toxicity was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. A slight increase in the number of revertant colonies was observed with TA100, in the absence of S9 metabolism, and with WP2uvrA tester strain, in the presence of S9 metabolic activation, only at the highest dose level. A more pronounced mutagenic effect was observed with TA100 tester strain in the presence of S9 metabolism, where a dose related increase in revertant colonies was noticed at higher dose levels.

Main Assay I

5000, 2500, 1250, 625 and 313 µg/plate, reductive metabolic activation system.

At the end of the incubation period, no precipitation of the test item neither toxic effects were observed with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Dose related increases in the number of revertant colonies were noticed with TA1537 and TA100 tester strains, both in the absence and presence of S9 metabolic activation. With TA1537, the revertant numbers were greater than twice the concurrent negative control, at the highest dose level.

Main Assay II

5000, 2500, 1250, 625 and 313 µg/plate, reductive metabolic activation system

No precipitation of the test item was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Severe toxicity was observed with TA98 and TA100 tester strains, at all dose levels, both in the absence and presence of S9 metabolism,while a slight toxic effect was noticed with TA1535, at the highest concentration tested, both in the absence and presence of S9 metabolic activation. Increases in revertant numbers were observed with TA1537 tester strain, both in the absence and presence of S9 metabolic activation, confirming the positive results obtained in the previous assay.

Main Assay III

TA1537-TA100, 5000, 3300, 2220, 1480 and 988 µg/plate, standard metabolic activation system.

No precipitation of the test item was observed, at the end of the incubation period, with any tester strain, at any dose level, in the absence or presence of S9 metabolism. Once again, treatments of TA1537 tester strain, both in the absence and presence of S9 metabolism, produced dose-related increases, which were greater than twice the control value at the highest dose level. Slight, but dose related and reproducible increases observed with TA100 tester strain, were al most certainly a further evidence of this mutagenic effect.

It is concluded that the test item induces reverse mutation in Salmonella typhimurium, both in the absence and presence of S9 metabolism, under the reported experimental conditions.

 

 

In vitro gene mutation study in mammalian cells - MUTATION IN L5178Y TK+/ − MOUSE LYMPHOMA CELLS (FLUCTUATION METHOD) SCREENING TEST

 

The test item was examined for mutagenic activity by assaying for the induction of 5 trifl uorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.

A main experiment was performed using a short treatment time (3 hours), both in the absence and presence of S9 metabolism and a long treatment time (24 hours) in its absence. Test item solutions were prepared using Complete Medium. The test item was assayed at a maximum dose level of 2000 µg/mL (the upper limit to testing indicated in the Study Protocol) and at the following dose levels: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 µg/mL.

In the absence of S9 metabolism, using the short treatment time, no cells survived treatment at the four highest dose levels. At the next two lower concentrations of 125 and 62.5 µg/mL, the Relative Total Growth (RTG) was reduced to approximately 37%. Using the long treatment time, no cells were recovered at the end of treatment at the three highest concentrations.

The next lower concentration (250 µg/mL) yielded severe toxicity (2% RTG), while dose related toxicity was noted over the remaining concentrations tested reducing RTG to 19% at 125 µg/mL. In the presence of S9 metabolism, no cells were recovered after treatement at the four highest concentrations, while dose related toxicity, from severe (6% RTG) to slight (68% RTG), was noted at the remaining dose levels.

Increases in mutant frequency were observed following treatment with the test item, both in the absence and presence of S9 metabolism. Using the short treatment time, both in the absence and presence of S9 metabolism, the Induced Mutation Frequency (IMF) was lower than the Global Evaluation Factor (GEF) at all concentrations tested. However, a statistically signifi can't increase in mutation frequency over the concurrent negative control was observed at higher analysable concentrations and results exceeded the negative historical control range. Using the long treatment time, an increase higher than the GEF was noted at the highest analysable concentration. Moreover, mutation frequencies at the next two lower dose levels fell out the negative historical control range. A statistically significant dose-effect relationship was observed in all treatment series.

It is concluded that the substance induced mutation at the TK locus of L5178Y mouse lymphoma cells cultured in vitro, under the reported experimental conditions.

 

Following the indications inside the " Guidance on Information Requirementsand Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance" (V 6.0 July 2017): Mutagenicity testing strategy ( Figure R.7.7–1 Flow chart of the mutagenicity testing strategy)

1) REACH Annex VII: Ames test has been conducted: positive

2) REACH Annex VIII: Mouse lymphoma assy has been conducted: positive

3) REACH Annex IX: comet assay test is under development

 

Standard information requirement at Annex VII: A preliminary assessment of mutagenicity is required for substances at the REACH Annex VII tonnage level. All available information should be included but, as a minimum, there should normally be data from a gene mutation test in bacteria unless existing data for analogous substances indicates this would be inappropriate.

When the result of the bacterial test is positive, it is important to consider the possibility of the substance being genotoxic in mammalian cells. The need for further test data to clarify this possibility at the Annex VII tonnage level will depend on an evaluation of all the available information relating to the genotoxicity of the substance.

 

According to the Figure R.7.7–1 Flow chart of the mutagenicity testing strategy, described in the "Guidance on Information Requirementsand Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance" (V 6.0 July 2017), in case a positive result is obtained from the in vitro gene mutation study in bacteria, it is needed to proceed with ANNEX VIII. Further evaluation of the mutagenic potential of the substance was therefore done by following OECD 490 In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene, in order to evaluate the gene mutation in mammalian cells.

Following the same figure ( R.7.7–1) due to a positive result obtained in the mouse lymphoma assay (tk +/-locus) a further investigation is needed (as per ANNEX IX) and thus the Comet assay (OECD guideline 489 In Vivo Mammalian Alkaline Comet Assay) performed on a Similar substance, currently in progress, will be used.

Justification for classification or non-classification

Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of the genetic material of cells or organisms. These changes may involve a single gene or gene segment, a block of genes or chromosomes. The term clastogenicity is used for agents giving rise to structural chromosome aberrations. A clastogen can cause breaks in chromosomes that result in the loss or rearrangements of chromosome segments.

Aneugenicity (aneuploidy induction) refers to the effects of agents that give rise to a change (gain or loss) in chromosome number in cells. An aneugen can cause loss or gain of chromosomes resulting in cells that have not an exact multiple of the haploid number. For example, three number 21 chromosomes or trisomy 21 (characteristic of Down syndrome) is a form of aneuploidy.

Genotoxicity is a broader term and refers to processes which alter the structure, information content or segregation of DNA and are not necessarily associated with mutagenicity. Thus, tests for genotoxicity include tests which provide an indication of induced damage to DNA (but not direct evidence of mutation) via effects such as DNA strandbreaks, unscheduled DNA synthesis (UDS), sister chromatid exchange (SCE), DNA adduct formation or mitotic recombination, as well as tests for mutagenicity.  

In the risk assessment of substances it is necessary to address the potential effect of mutagenicity with an in vivo test.

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny.

Substance that are mutagenic in somatic cells may produce heritable effects if they, or their active metabolites, have the ability to interact with the genetic material of germ cells. Conversely, substances that do not induce mutations in somatic cell in vivo would not be expected to be germ cell mutagens.

However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

Positive results are obtained from the studies In vitro gene mutation study in bacteria (AMES) and In vitro gene mutation study in mammalian cells conducted on the substance. In order to conclude on the classification of the substance an in vivo study is required; a Comet assay (OECD guideline 489 In Vivo Mammalian Alkaline Comet Assay) performed on a Similar substance, is currently in progress. Without a complete assessment there are no sufficient elements to determine the mutagenicity of the test item, therefore the classification according to the CLP Regulation ( EC 1272/2008) is inconclusive until the finalisation of the In Vivo Mammalian Alkaline Comet Assay test.