9,10-Anthracenedione, 1,4-bis[[3-[2-(2-hydroxyethoxy)ethoxy]propyl]amino]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO Negative (with and without metabolic activation); bacterial reverse mutation assay in S. typhimurium; OECD 471; EU Method B.13/14 Negative (with and without metabolic activation); in vitro mammalian cell micronucleus test in human lymphocytes; OECD 487

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:

4 November 2004 to 10 December 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA EPS (TSCA) OPPTS harmonised guidelines

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(the method conforms with those published by METI, MHLW and MAFF)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

10 % liver S9 in standard co-factors

Test concentrations with justification for top dose:

- Preliminary Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Mutation Test (experiments 1 and 2): 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water. Formulated concentrations were adjusted to make allowances for the purity of the test material.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene, 1,8-dihydroxyanthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);
A measured aliquot (0.1 mL) of bacterial culture was dispensed into a tube followed by 2.0 mL of molten, trace histidine-supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9 mix or phosphate buffer. The content of the tube was dispensed onto the surface of a Vogel-Bonner minimal agar plate and incubated.

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: The procedure was repeated in triplicate for each concentration in each bacterial strain for both Experiment 1 and Experiment 2.

NUMBER OF CELLS EVALUATED: The frequency of revertant colonies was assessed using a Domino colony counter. The top dose level was assessed manually due to test material-induced colouration.

DETERMINATION OF CYTOTOXICITY
- Method: Assessment of effects on the growth of the background bacterial lawn.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met:
- the test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count of at least one strain of bacteria.

Statistics:

Dunnett’s method of linear regression was used to assess statistical significance.

Species / strain:

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

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:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material caused no visible reduction in the growth of the background bacterial lawn at any dose level. A blue colour was observed at and above 50 µg/plate, becoming darker with increasing concentration. This did not prevent scoring of revertant colonies. No test material precipitate was observed on any of the plates at any of the dose levels tested in either the presence or the absence of S9 mix.
No significant increases in the frequency of revertant colonies were recorded for any of the strains at any dose level with or without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Experiment 1 Summary of Results

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
- - - - - -	0 50 150 500 1500 5000	132 148 140 144 146 132	31 30 32 29 20 19	304 339 350 303 353 338	23 20 27 25 16 20	15 18 12 16 13 12
+ + + + + +	0 50 150 500 1500 5000	134 120 138 132 105 125	16 10 10 11 12 9	374 377 345 283 376 374	37 42 44 41 28 28	19 17 20 19 17 18
Positive Controls
-	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentration (µg/plate)	3	5	0.5	0.2	80
	Mean no. colonies/plate	740	621	1213	164	629
+	Name	2AA	2AA	DAN	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	802	231	1029	245	437

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

MMC = mitomycin C

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

DAN = 1,8-dihydroxyanthraquinone

 

Table 2: Experiment 2 Summary of Results

+/- S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	TA102	TA98	TA1537
- - - - - -	0 50 150 500 1500 5000	88 78 86 77 90 72	20 19 14 17 19 15	371 348 364 341 258 335	17 15 12 17 18 12	14 20 15 12 10 13
+ + + + + +	0 50 150 500 1500 5000	79 87 92 93 85 78	11 12 11 11 10 12	352 354 349 348 343 360	28 37 36 29 34 20	19 15 23 16 14 17
Positive Controls
-	Name	ENNG	ENNG	MMC	4NQO	9AA
	Concentration (µg/plate)	3	5	0.5	0.2	80
	Mean no. colonies/plate	338	465	1108	135	490
+	Name	2AA	2AA	DAN	BP	2AA
	Concentration (µg/plate)	1	2	10	5	2
	Mean no. colonies/plate	566	233	905	254	376

ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine

MMC = mitomycin C

4NQO = 4-Nitroquinoline-1-oxide

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

BP = benzo(a)pyrene

DAN = 1,8-dihydroxyanthraquinone

Conclusions:

Interpretation of results (migrated information):
negative with or without metabolic activation

Under the conditions of this study, the test material was found to be devoid of mutagenic activity on Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and TA 102 in the presence and absence of metabolic activation.

Executive summary:

The mutagenic potential of the test material was investigated in a bacterial reverse mutation assay which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, USA EPS (TSCA) OPPTS harmonised guidelines and those published in Japan by METI, MHLW and MAFF under GLP conditions.

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA100 and TA 102 were treated with the test material (using the Ames plate incorporation method) at dose levels of 50, 150, 500, 1500 and 5000 µg/plate in distilled water both in the presence and absence of metabolic activation (S9-mix). Concurrent solvent, negative and positive controls were run.

The tets material caused no visible reduction in the growth of the background bacterial lawn at any dose level. A blue colour was observed at and above 50 µg/plate, becoming darker with increasing concentration. This did not prevent scoring of revertant colonies. No test material precipitate was observed on any of the plates at any of the dose levels tested in either the presence or the absence of S9 mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains at any dose level with or without metabolic activation.

Under the conditions of this study, the test material was found to be devoid of mutagenic activity on Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA 100 and TA 102 in the presence and absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Bacterial Reverse Mutation Assay

The mutagenic potential of the test material was investigated in a bacterial reverse mutation assay which was conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, USA EPS (TSCA) OPPTS harmonised guidelines and those published in Japan by METI, MHLW and MAFF under GLP conditions.

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA100 and TA 102 were treated with the test material (using the Ames plate incorporation method) at dose levels of 50, 150, 500, 1500 and 5000 µg/plate in distilled water both in the presence and absence of metabolic activation (S9-mix). Concurrent solvent, negative and positive controls were run.

The tets material caused no visible reduction in the growth of the background bacterial lawn at any dose level. A blue colour was observed at and above 50 µg/plate, becoming darker with increasing concentration. This did not prevent scoring of revertant colonies. No test material precipitate was observed on any of the plates at any of the dose levels tested in either the presence or the absence of S9 mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains at any dose level with or without metabolic activation.

Under the conditions of this study, the test material was found to be devoid of mutagenic activity on Salmonella typhimurium strains TA 1535, TA 1537, TA 98 TA100 and TA 102 in the presence and absence of metabolic activation.

In Vitro Mammalian Cell Micronucleus Test

The test material was evaluated in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in two main experiments in accordance with the standardised guideline OECD 487 under GLP conditions.

Cells were exposed to the test material in the 3 + 21 hour treatments (with and without metabolic activation) at concentrations of 0, 250, 500, 1000, 2000, 3000, 4000 and 5000 µg/mL. Cells were exposed to the test material in the 24 + 0 hour treatment (without metabolic activation) at concentrations of 0, 50, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 500 and 600 µg/mL.

The test material was formulated in DMSO, and appropriate solvent and positive controls were run concurrently. Treatments were conducted 48 hours following mitogen stimulation by Phytohaemagglutinin (PHA). In Experiment 1, micronuclei were analysed at three or four concentrations. The data for the 3+21 hour treatment in the presence of S-9 in Experiment 1 were considered inconclusive; therefore a further experiment was performed under these treatment conditions. In Experiment 2, micronuclei were analysed at three concentrations.

Treatment of cells with the test material for 3+21 hours and 24+0 hours in the absence of S-9 resulted in frequencies of binucleate cells with micronuclei (MNBN) that were not significantly different from those observed in concurrent vehicle controls at any concentration analysed. The MNBN cell frequency of all treated cultures fell within the normal range.

Treatment for 3+21 hours in the presence of S-9 in Experiment 1 resulted in frequencies of MNBN cells that were significantly higher (p≤0.001) than those observed in concurrent vehicle controls at the highest concentration analysed (5000 μg/mL), but not at 3000 and 4000 μg/mL. The MNBN cell frequency of treated cultures fell within the upper limit of the 95th percentile of the normal range at 3000 and 4000 μg/mL but both cultures exceeded this range at 5000 μg/mL. However, the MNBN cell frequency in one of the two cultures at 5000 μg/mL fell only marginally outside the normal range and there was marked heterogeneity. These data were considered inconclusive.

Treatment for 3+21 hours in the presence of S-9 in Experiment 2 resulted in frequencies of MNBN cells that were not significantly different from those observed in concurrent vehicle controls at all concentrations analysed (3000, 4000 and 5000 μg/mL). All MNBN cell frequencies in treated cultures fell within the normal range.

Some differences in toxicity were seen between experiments at 5000 μg/mL (41 and 11 % reductions in RI were seen in Experiments 1 and 2, respectively), but no concentration achieved 50 % toxicity in either experiment.

Overall, there was poor reproducibility between experiments at 5000 μg/mL in terms of toxicity and induction of micronuclei. Due to the lack of reproducibility between experiments, these data do not indicate evidence of micronucleus induction in the presence of S-9.

Under the conditions of this study, the test material did not induce increases in the frequency of micronuclei in the absence and presence of metabolic activation (S-9) when tested up to 5000 μg/mL.

Justification for selection of genetic toxicity endpoint
The study was conducted under GLP conditions in accordance with standardised guidelines and was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). This study was selected as key on the basis that it is one of the standard information requirements for this level of notification.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to genetic toxicity.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to genetic toxicity.