Dinaphtho[1,2,3-cd:3',2',1'-lm]perylene-5,10-dione, (9,10-dihydro-9,10-dioxo-1-anthracenyl)amino derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In the key study selected, the test was performed to investigate the potential of FAT 45082/G to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 according to OECD test guideline 471. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate. No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with FAT 45082/G at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. 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.

In a supporting study, FAT 45082/A was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the concentrations of 25, 75, 225, 675 and 2025 ug/ml without and with microsomal activation. No evidence of the induction of point mutations by FAT 45082/A or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, FAT 45082/G is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November, 1995 to 29 January, 1996

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:

only 4 tester stains used; a tester strain to detect cross-linking mutagens was not included

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Code number: FAT 45082/G
- Batch-Nr.: Op. 11.56
- Purity: ca. 85 %
- Appearance: solid, black
- Solubility: insoluble [in water]
- Storage: room temperature
- Expiration date: 10/1999

Target gene:

Histidine auxotrophs

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

None

Additional strain / cell type characteristics:

other: The Salmonella typhimurium histidine (his) reversion system measures his- —> his+ reversions.

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua bidest and DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative nontoxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

aqua dest.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535, TA 100; Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537, TA 98; Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

TA 1535, TA 1537, TA 98, TA 100; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Mammalian Microsomal Fraction S9 Mix
The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

S9 (Preparation by CCR)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf, weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously. After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, was diluted 1+3 in KCl and centrifuged at 9,000 g for 10 minutes at 4° C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at - 20 °C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 0006. The protein concentration in the S9 preparation was 32.4 mg/ml (lot 271195).

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was chosen to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Pre-Experiment for Toxicity
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment: were the same as described for the experiment I below (plate incorporation test). Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Experimental Performance
For each strain and dose level, including the controls three plates were used as a minimum.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl : Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 ul : S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µl: Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µl: Overlay agar

In the pre-incubation assay 100 µl test solution, 500 ul S9 mix / S9 mix substitution buffer and 100 ul bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

According to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537.
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 criteria described above or not.

Statistics:

No data

Key result

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:

not examined

Positive controls validity:

valid

Additional information on results:

None

None

Conclusions:

FAT 45'082/G is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of FAT 45'082/G to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500; and 5000 µg/plate

In experiment I and II, toxic effects, evidenced by a reduction in the number of revertants, occurred in the strains TA 1535 and TA 1537 at the higher concentrations with and without metabolic activation.

The background growth was reduced at 2500 and 5000 ug/plate with and without S9 mix in both experiments.

No substantial increases in revertant colony numbers of any of the four tester strains were observed following treatment with FAT 45'082/G at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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, FAT 45'082/G is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

FAT 45082 is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assays, hence it does not warrant classification as per the criteria of Regulation (EC) no. 1272/2008 (CLP).