N,N'-[6,13-diacetamido-2,9-diethoxy-3,10-triphenodioxazinediyl]bis(benzamide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacteria:

OECD 471; GLP; S.typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2 uvrA; 33 -5000 µg/plate; no cytotoxicity; non-mutagenic

Cytogenicity:

OECD 473; GLP; human lymphocytes; 40 -150 µg/ml; no cytotoxicity; not clastogenic

Gene mutation:

OECD 476; GLP; mouse lymphoma; 62.5 -2000 µg/ml; no cytotoxicity; non-mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- 98.3%

Species / strain / cell type:

lymphocytes: human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments

Details on mammalian cell type (if applicable):

Blood from three healthy, non-smoking male volunteers from a panel of donors was used for each experiment. No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication. HEPES-buffered RPMI medium

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 mix

Test concentrations with justification for top dose:

Experiment 1:
+ S9 mix: 0, 40, 60, 80 ug/ml
- S9 mix: 0, 80, 100, 150 ug/ml

Experiment 2:
+ S9 mix: 0, 40, 50, 55 ug/ml
- S9 mix: 0, 45, 60, 75, 90 ug/ml

Vehicle / solvent:

DMF

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h +/- S9 mix experiment I, 20h without S9 mix and 3h with S9 mix experiment II
- Expression time (cells in growth medium): after 3h exposure 17h, after 20h exposure 0h
- Fixation time (start of exposure up to fixation or harvest of cells): after 4h exposure 18-28h, 18h and 28h exposure and fixation direct thereafter

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments, samples / cultures in triplicate

NUMBER OF CELLS EVALUATED: 100 well-spread metaphases will be scored per culture

DETERMINATION OF CYTOTOXICITY
- Method: slides will be evaluated for mitotic index and cell numbers, 1000 cells per culture will be scored and values will be expressed as a percentage of the solvent controls

Evaluation criteria:

For valid data, the test article was considered to induce clastogenic events if:
1. A proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p0.05)
3. There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered positive in this assay if all of the above criteria were met.

Statistics:

The statistical method used was Fisher's exact test. The proportions of aberrant cells in each replicate were also used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test.

Species / strain:

lymphocytes: Human Lymphocyte

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation was observed at 55 and 150 ug/ml

Conclusions:

It is concluded that the test substance did not induce chromosome aberrations in cultured human peripheral blood lymphocytes following treatment in the absence and presence of rat liver metabolic activation system (S-9). Concentrations were tested up to the limit of solubility within the test system. Therefore, the test substance was considered to be non-clastogenic in this in vitro test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Purity: 98.3%

Target gene:

HPRT locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media supplied containing L-glutamine and HEPES
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Acroclor induced liver S9 mix

Test concentrations with justification for top dose:

Experiment 1 ten concentrations, ranging from 50 to 1000 µg/mL in the absence of S-9 and from 25 to 625 µg/mL in the presence of S 9 mix
Experiment 2 ten concentrations, ranging from 50 to 750 µg/mL in the absence of S 9 and from 25 to 500 µg/mL in the presence of S-9 mix

Vehicle / solvent:

DMF

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h
- Expression time (cells in growth medium): 7 days at 37°C
- Selection time (if incubation with a selection agent): 6-7 days in TG-medium
- Fixation: At the end of the selection period, the medium will be removed and the remaining colanies will be fixed with methanol, stained with Giemsa and counted

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the vehicle control (p0.05)
2. There was a significant concentration relationship as indicated by the linear trend analysis (p0.05)
3. The effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No marked changes in osmolality or pH. Precipitation was observed at the highest concentrations.

Conclusions:

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to precipitating concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9). Therefore, the test substance is considered to be non-mutagenic in this in vitro test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The protein concentration in the S9 preparation was 47.3 mg/mL and not in the range of 20 - 45 mg/mL as stated in the protocol.

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Purity: 99%

Target gene:

his-locus

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

strains TA 1535 and TA 100, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

strains TA 1537 and TA 98, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

strain WP2 uvrA, without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains, with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment I); preincubation (Experiment II)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

- A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
- A test article producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.

- A test article is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
- Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation of the data is required.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The background growth was reduced at 2500 and 5000 µg/plate with and without metabolic activation in both independent experiments.
No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia Coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant study, performed according to OECD guideline 471, the test substance was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) at concentrations up to 5000 ug/plate using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

The test item was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. Treatment of cells with in the absence and presence of S-9 in Experiments 1 and 2 resulted in frequencies of cells with structural chromosome aberrations that were similar to those observed in concurrent vehicle control cultures for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges. Therefore,the test material was considered to be non-clastogenicin this in vitro test system.

The test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S‑9). In Experiments 1 and 2 when tested up to precipitating concentrations, no statistically significant increases in mutant frequency, compared to the concurrent vehicle controls, were observed following treatment at any concentration analysed in the absence and presence of S‑9 and there were no statistically significant linear trends. Therefore,the test item is considered to be non-mutagenicin this in vitro test system.

Short description of key information:
The test substance was unable to induce mutation in five strains of Salmonella typhimurium when tested up to 5000 µg/plate in the absence and presence of a rat liver metabolic activation system.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.