Chromium, 1-[[2-hydroxy-4(or 5)-nitrophenyl]azo]-2-naphthalenol complex

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance is not mutagenic in mammalian cells, as determined in an OECD 476 study (Stahl, 2016).

The test substance is not chromosome damaging, as determined in an OECD 487 study (Stahl, 2016).

OECD 471, nitro-reducatse deficient strains, marked reduction in mutagenic activity

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read across from analogue substance

Adequacy of study:

weight of evidence

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

nitroreductase-deficient strains TA98NR and TA100NR

Principles of method if other than guideline:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA98NR and TA100NR

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from uninduced male Golden Syrian hamsters. The S-9 was stored frozen at <-50°C, and thawed prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 30% reductive (Prival) S-9 mix respectively. The composition of the mix and buffer solution are described in the following table:

Ingredient Final concentration/ml in:
30% reductive S9 mix Bufer solution
Sodium phosphate buffer pH 7.4 (SPB) 100 μmoles 100 μmoles
Glucose-6-phosphate (disodium) (G-6-P) 20 μmoles -
NADP(disodium) 4 μmoles -
NADH 2 μmoles -
Flavin mononucleotide (FMN) 2 μmoles -
Glucose-6-phosphate dehydrogenase 30 units -
Magnesium chloride (MgCl2) 8 μmoles -
Potassium chloride (KCl) 33 μmoles -
Water To volume To volume
S-9 (uninduced hamster liver) 300 μL -

Test concentrations with justification for top dose:

Mutation experiment:
2.5, 8, 25, 80, 250, 800 and 2500 µg/plate in the presence of a modified (reductive) S 9 mix
0.125, 0.4, 1.25, 4, 12.5. 40, 125, 400 and 1250 µg/plate in the absence of S-9.


The maximum treatment concentrations were limited by solubility of the test article in the primary vehicle, DMSO, and in the absence of S-9 also by the maximum volume additions that could be employed due to apparent vehicle-related toxicity. Following these treatments, evidence of toxicity was observed in all strains in the absence and presence of S-9. These toxic effects were observed at the highest treatment concentration of 1250 µg/plate in all strains except TA102 in the absence of S-9, and in strain TA102 and all strains in the presence of S-9 the toxic effects extended down to differing concentrations in each case that were between 8 and 800 µg/plate. The most extensive toxicity was observed in strains TA1535 and TA1537 in the presence of S-9, where only one or two non-toxic treatment concentrations remained. As mutation data were therefore available from fewer than 5 analysable concentrations for these strain treatments, these were repeated to provide a more thorough and robust assessment of the mutagenicity of the test article in this assay system

Vehicle / solvent:

anydrous analytical grade DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

congo red

mitomycin C

other: Metronidazole, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in a pre-incubation methodology
- the experiment is performed in triplicate

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 minutes
- Exposure duration/duration of treatment: 3 days, plasted inverted and protected from light


Treatments were performed using a pre-incubation methodology in the absence and presence of a modified (reductive) S-9 mix. These platings were achieved by the following sequence of additions to sterile pre incubation tubes:
• 0.1 mL of bacterial culture
• 0.1 mL of test article formulation/vehicle control or 0.05 mL of positive control
• 0.5 mL of 30% reductive S-9 mix or buffer solution.
Quantities of test article formulation or control solution, bacteria and S-9 mix or buffer solution detailed above, plus an additional 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4), were mixed together and placed in an orbital incubator set to either 37C (for the treatments in the absence of S-9) or 30C (for treatments in the presence of S 9) for 30 minutes, before the addition of 2 mL of supplemented molten agar at 45±1°C followed by rapid mixing and pouring on to Vogel-Bonner E agar plates.
When set, the plates were inverted and incubated protected from light for 3 days in an incubator set to 37C. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted

The addition of 0.5 mL of 100 mM sodium phosphate buffer (pH 7.4) to these Mutation Experiment treatments was employed to reduce the solvent concentration during the pre-incubation period. DMSO, and some other organic solvents, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By employing the modification indicated, the DMSO concentration in the pre-incubation mix was decreased, in an attempt to minimise or eliminate any toxic effects of the solvent that may have otherwise occurred. In order to ‘correct’ for the additional volume in the pre-incubation mix, these were plated out using 2 mL of 1.125% top agar (rather than the 0.9% top agar used for ‘standard’ pre incubation treatments), therefore the additions to each plate were comparable to that of the other pre-incubation treatments.

It should be noted that data from the initial treatments of all the tester strains in the absence and presence of S-9 were invalidated, as the plates demonstrated evidence of vehicle-related toxicity, and toxic effects extended over almost all treatment concentrations (considered to have been exacerbated by the vehicle-related toxicity). Repeat treatments were performed (using the treatment methodology described above) with all strains in the absence and presence of S-9. The data from these repeat treatments in the presence of S-9 were considered acceptable (and are those presented as the Mutation Experiment data for each strain in the presence of S-9), but the data from the test article treatments in the absence of S-9 were again invalidated, due to toxic effects extending over almost all treatment concentrations, and evidence of vehicle-related toxicity.
Further repeat treatments of all strains in the absence of S-9 were therefore performed. The methodology was amended slightly in order to address the apparent vehicle-related toxicity. Vehicle and test article treatments were performed using volume additions reduced to 0.05 mL, and the further 0.5 mL addition of 100 mM sodium phosphate buffer (pH 7.4) was therefore not required, and consequently following the pre-incubation period, these treatments were mixed with 2 mL of supplemented 0.9% molten (top) agar and poured onto Vogel-Bonner E agar plates. These plates were then incubated and scored as indicated above.
Due to data from fewer than 5 analysable concentrations being available from the first repeat treatments of strains TA1535 and TA1537 in the presence of S-9, further treatments of these strains were also performed alongside the further repeat treatments in the absence of S-9, and employed the same modified treatment methodology (0.05 mL volume additions, no additional 100 mM sodium phosphate buffer (pH 7.4) added and plate out using 0.9% molten top agar). The results from these treatments are reported as the Mutation Experiment further treatments data.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as intensely coloured agar or bubbles or splits in the agar affected the accuracy of the automated counter.

- OTHER:

Rationale for test conditions:

The objective of this study was to evaluate the ability of test item to induce reverse mutations in histidine-requiring strains of Salmonella typhimurium in the absence and presence of a reductive hamster liver metabolising system (S-9). By assessing the mutagenicity of test item in nitroreductase deficient strains (TA98NR and TA100NR, the tow commercially available at the time of testing) alongside parent nitroreductase competent strains (TA98 and TA100), the role of nitroreduction in any test article related mutagenic activity could be determined. As the test substance is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
The test article was considered positive in this assay if the above criterion was met.
The test article was considered negative in this assay if the above criterion was not met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations.
Data from strain TA98 were compared (non-statistically) with that from TA98NR, and data from strain TA100 were compared with that from TA100NR. Where a mutagenic response was seen in one or both parent strains but was absent or much reduced in the corresponding NR variant strain(s), this was considered to be indicative that bacterial nitroreduction enzymes play a significant role in the mutagenicity of the test compound as observed in this study.

Statistics:

Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges (see Attachments).
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example cytotoxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of
critical importance.

Species / strain:

bacteria, other: TA 98NR

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

except at the highet tested dose of 1250 ug/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: TA100 NR

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

bacteria, other: TA100 NR

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

except at the highest tested dose of 2500 ug/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Following test item treatments of all the test strains in the absence and presence of S-9, clear and concentration-related (in some cases up to the toxicity and/or precipitating range) increases in revertant numbers were observed in strains TA98 and TA98NR in the absence and presence of S-9, in strain TA1537 in the absence of S-9, and in strains TA100, TA100NR and TA102 in the presence of S-9 In each case, these increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strain TA1537) the concurrent vehicle control values, although it should be noted that in strains TA100 and TA100NR in the presence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 2500 µg/plate

for the tables of the number of revertants per strain with and without metabolic, refer to the attached background information

Conclusions:

The analogue substance was tested for gene mutation in bacteria following OECD 471 with nitroreductase deficinet strains. Under the experimental conditions the mutagenicity of the substance was reduced for the NR strains confirming that direct acting mutagenicity of the substances, based on the nitroreductase activity, plays a major role in the mutagenic properties of the substance

Executive summary:

The test substance was assayed for mutation in seven histidine-requiring strains (TA98, TA100, TA1535, TA1537, TA102, TA98NR and TA100NR) of Salmonella typhimurium, both in the absence and in the presence of a reductive hamster liver metabolising system (S-9) in a single experiment.

All test item treatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO). As the test item is an azo compound, testing in the presence of S-9 in this study was performed using a modified reductive (Prival) S-9 pre-incubation methodology, as it is known that azo compounds can be reduced to free aromatic amines, which can be mutagenic.

Mutation Experiment treatments of the tester strains were performed using a pre‑incubation methodology using final concentrations of test substance at 2.5, 8, 25, 80, 250, 800 and 2500 µg/plate in the presence of a modified (reductive) S‑9 mix or at 0.125, 0.4, 1.25, 4, 12.5. 40, 125, 400 and 1250 µg/plate in the absence of S-9. The maximum treatment concentrations were limited by solubility of the test article in the primary vehicle, DMSO, and in the absence of S-9 also by the maximum volume additions that could be employed due to apparent vehicle-related toxicity. Following these treatments, evidence of toxicity was observed in all strains in the absence and presence of S-9. These toxic effects were observed at the highest treatment concentration of 1250 µg/plate in all strains except TA102 in the absence of S-9, and in strain TA102 and all strains in the presence of S-9 the toxic effects extended down to differing concentrations in each case that were between 8 and 800 µg/plate. The most extensive toxicity was observed in strains TA1535 and TA1537 in the presence of S-9, where only one or two non-toxic treatment concentrations remained. As mutation data were therefore available from fewer than 5 analysable concentrations for these strain treatments, these were repeated to provide a more thorough and robust assessment of the mutagenicity of the test article in this assay system.

The Mutation Experiment further treatments of strains TA1535 and TA1537 in the presence of a modified (reductive) S‑9 mix were performed using final concentrations of the test item at 0.125, 0.4, 1.25, 4, 12.5, 40, 125, 400 and 1250 µg/plate. Although the treatment concentrations were not markedly reduced compared to those previously described, a slightly modified methodology was employed (as was used for the treatments used to provide the Mutation Experiment data for all strains in the absence of S-9), whereby the test article and vehicle volume additions were reduced, as it was considered that a vehicle-related effect was exacerbating the observed test article-related toxicity. Following these treatments, no clear evidence of toxicity was observed.

Precipitation of test article was observed on all of the test plates treated at 250 µg/plate and above in each experiment.

Vehicle and positive control treatments were included for all strains in each experiment. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following test item treatments of all the test strains in the absence and presence of S-9, clear and concentration-related (in some cases up to the toxicity and/or precipitating range) increases in revertant numbers were observed in strains TA98 and TA98NR in the absence and presence of S-9, in strain TA1537 in the absence of S-9, and in strains TA100, TA100NR and TA102 in the presence of S-9. In each case, these increases were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98, TA98NR, TA100 or TA100NR) or ≥3-fold (in strain TA1537) the concurrent vehicle control values, although it should be noted that in strains TA100 and TA100NR in the presence of S-9, the 2-fold threshold level was only achieved at the maximum treatment concentration of 2500 µg/plate.

The mutagenic response in the nitroreductase deficient strain TA98NR in the absence of S-9 was reduced in magnitude compared to that with the corresponding treatments in the parent (nitroreductase proficent) strain TA98. Comparison of the magnitude of responses in these two strains in the presence of S-9 suggested that they were comparable, when assessed in terms of fold increase compared to the concurrent control level. However, the vehicle control counts in strain TA98NR were much smaller (mean of 19.3 revertants/plate) compared to that in strain TA98 (mean of 53.3 revertants/plate). Although the vehicle counts in each strain were considered acceptable, the much smaller vehicle control counts in strain TA98NR served to exaggerate the relativeAcid Black 63:2response when assessed in terms of fold increase. When the mean number of induced revertants/plate were assessed, the largest increase in strain TA98 was 176 revertants/plate (229.3 – 53.3), as compared to the largest increase in strain TA98NR of just 79 revertants/plate (98.3 – 19.3), which indicated a reduced mutagenic response in the nitroreductase deficient strain TA98NR. When the mutagenicity in strains TA100 and TA100NR were compared, there were similar mutagenic responses observed in both strains in the presence of S‑9, but in the absence of S-9 there was no response seen in strain TA100NR compared to a weak mutagenic response observed in strain TA100. Although less clear than the differential between strains TA98 and TA98NR, and only apparent for the treatments in the absence of S-9, these data indicated a reduced mutagenic response in the nitroreductase deficient strain TA100NR. Overall, the data from strains TA98, TA98NR, TA100 and TA100NR were considered to indicate that nitroreduction does play a significant role in the mutagenic activity of the test item seen in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

HPRT assay

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro in a GLP compliant study according to OECD Guideline 476, EU Method B. 17 and EPA OPPTS 870.5300 (BASF SE, 2016). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). As follow-up on the revision of the OECD Guideline No. 476 minor changes in test procedure were implemented in this study (e.g. increased numbers of seeded cells and enzymatic dissociation of the cells at the end of exposure period). Although validated inhouse no robust dataset on this setup recently exist, these changes may have a minor impact on the data. However, it was concluded to use for data interpretation of this study the current historical control database obtained in the period from January 2013 to December 2014.

In the 1st experiment the following concentrations were tested with and without S9 mix: 0; 2.5; 5.0; 10.0; 20.0; 40.0; 80.0; 160.0 μg/mL. In the 2nd experiment the following concentrations were tested with and without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0; 160.0 μg/mL. The 6.3 μg/mL was not tested for gene mutations.

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected increase in the frequencies of forward mutations.

In this study in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Based on the results of the present study, the test substance did not cause any statistically significant and dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Micronucleus Assay

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) in a GLP compliant study according to OECD Guideline 487 and EU method B. 49 (BASF SE, 2016). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested.

1st 4 hours exposure, 24 hours harvest time, without S9 mix

- Tested concentrations: 0; 5.00; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups: 0; 40.00; 80.00; 160.00 μg/mL

1st Experiment_4 hours exposure, 24 hours harvest time, with S9 mix

- Tested concentrations: 0; 5.00; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups: 0; 5.00; 10.00; 20.00μg/mL

2nd Experiment, 24 hours exposure, 24 hours harvest time, without S9 mix

- Tested concentrations:0; 10.00; 20.00; 40.00; 80.00; 160.00 μg/mL

- Evaluated test groups:0; 40.00; 80.00; 160.00 μg/mL

2nd Experiment, 4 hours exposure, 44 hours harvest time, with S9 mix

- Tested concentrations: 0; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL

- Evaluated test groups: 0; 5.00; 10.00; 20.00 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

No cytotoxicity, indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI), was observed up to the highest applied test substance concentration. The highest applied test substance concentration was clearly precipitating in cell culture medium under all experimental conditions.

On the basis of the results of this study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.

Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

General conclusion

the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation and non-mutagenic in the in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Kirkland et al. (Mutation Research 775-776 (2014) 69-80) published an article in which the authors conclude that: “Thus, in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential."

Therefore, the test item is not considered to induce genotoxicity and the presented studies and data are sufficient evidence to waive the need for in vivo gene mutation testing.

Justification for classification or non-classification

Classification for mutagenicity is warranted for substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases

from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by

positive results from in vitro mutagenicity assays.

Based on the results of the in vitro tests no classification for mutagenicity is applied following Regulation 1272/2008.