1,4-bis(butylamino)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material showed mutagenic effects in 1 strain of Salmonella typhimurium (TA 98) with metabolic activation.

The test item did not induce gene mutations inthe hprt locus in CHO cells in vitro.

Link to relevant study records

Reference

Reference 1

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:

2014-08-26 till 2014-08-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: Better than others

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in strain TA 98 with metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Water solubility: unsoluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed
The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal back¬ground growth up to 5000 µg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol¬ic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Study Name: 1652800	Study Code: Harlan CCR 1652800
Experiment: 1652800 VV Plate	Date Plated: 26/08/2014
Assay Conditions:	Date Counted: 29/08/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			26 ± 4^B M	7 ± 2	21 ± 7	174 ± 10	35 ± 13
	Untreated			28 ± 3^B M	10 ± 3	22 ± 6	162 ± 30	48 ± 5
	Fat Blue B 01	3 µg		27 ± 4^B M	10 ± 4	28 ± 1	177 ± 2	42 ± 0
		10 µg		26 ± 2^B M	8 ± 2	19 ± 4	166 ± 9	39 ± 5
		33 µg		30 ± 2^B M	8 ± 1	22 ± 3	191 ± 2	44 ± 6
		100 µg		26 ± 1^B M	7 ± 1	17 ± 2	216 ± 16	41 ± 4
		333 µg		28 ± 2^B M	9 ± 1	19 ± 9	185 ± 16	54 ± 9
		1000 µg		28 ± 1^B D M	7 ± 1^D	21 ± 1^D	173 ± 10^D	44 ± 9^D
		2500 µg		26 ± 5^B D M	6 ± 2^D M	21 ± 4^D M	174 ± 11^D M	32 ± 4^D M
		5000 µg		28 ± 2^B D M	6 ± 1^D M	17 ± 2^D M	176 ± 13^D M	27 ± 4^D M
	NaN3	10 µg		2459 ± 235			1835 ± 57
	4-NOPD	10 µg				395 ± 13
	4-NOPD	50 µg			80 ± 8
	MMS	2.0 µL						1107 ± 49
With Activation	DMSO			13 ± 6	15 ± 2	30 ± 10	123 ± 23	55 ± 4
	Untreated			14 ± 7	12 ± 5	38 ± 6	175 ± 4	57 ± 14
	Fat Blue B 01	3 µg		19 ± 5	20 ± 1	41 ± 1	155 ± 7	41 ± 4
		10 µg		18 ± 2	18 ± 5	32 ± 3	146 ± 5	54 ± 6
		33 µg		17 ± 0	19 ± 4	37 ± 13	155 ± 4	53 ± 2
		100 µg		16 ± 4	16 ± 1	42 ± 2	163 ± 16	51 ± 5
		333 µg		13 ± 5	22 ± 4	73 ± 3	148 ± 16	50 ± 2
		1000 µg		14 ± 3^D	20 ± 5^D	117 ± 13^D	151 ± 18^D	46 ± 5^D
		2500 µg		13 ± 2^D M	21 ± 2^D M	172 ± 33^D M	168 ± 6^D M	49 ± 10^D M
		5000 µg		13 ± 3^D M	18 ± 2^D M	192 ± 7^D M	179 ± 5^D M	34 ± 3^D M
	2-AA	2.5 µg		454 ± 13	310 ± 18	2167 ± 35	4272 ± 129
	2-AA	10.0 µg						212 ± 10

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	D M B	Densely coloured plate Manual count Extensive bacterial growth

 

Conclusions:

Interpretation of results (migrated information):
positive in strain TA 98 with metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix.

Executive summary:

The test item Fat Blue B 01 was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmo­nella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and theEscheri­chia colistrain WP2 uvrA.

The assay was performed with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. No precipitation of the test item was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with Fat Blue B 01 at any dose level in the absence of metabolic activation (S9 mix).

In the presence of metabolic activation a substantial and dose dependent increase was observed in strain TA 98.The number of colonies exceeded the threshold of twice the number of the corresponding solvent control at concentrations from 333 µg/plate to 5000 µg/plate

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

The data in the untreated control of strain TA 1535 were slightly above our historical control range in the absence of metabolic activation. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

 

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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

Type of assay:

mammalian cell gene mutation assay

Target gene:

This mammalian cell mutation assay system detects point mutations involving base substitutions, deletions, frameshifts and rearrangements within the locus. The enzyme hypoxanthine guanine phosphoribosyl transferase (hprt) catalyses phosphorylation of purines in one of the purine salvage pathways. The selective agent used in this assay, 6-Thioguanine (6-TG), is also a substrate for this enzyme and cells that retain the functional hprt enzyme are susceptible to the cytotoxic effects of 6-TG. Forward mutations that result in the loss of the functional hprt gene render cells resistant to 6-TG. These mutant cells can be quantitated after an expression period by cloning in culture medium supplemented with 6-TG, the selective agent.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

A) 250 B) 500 C) 1000 and D) 2000 µg/mL (factor of 2)

Vehicle / solvent:

DMSO was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

- Source of the Test System:
American Type Culture Collection, P. O. Box 1549, Manassas, VA 20108, USA

- Storage of Test System
Stock cultures of the CHO-K1 cell line were stored in the test facility as frozen permanents in liquid nitrogen.

Evaluation criteria:

- Evaluation and Interpretation : When all the validity criteria are fulfilled:

a.A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:

•At least one of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•The increase is dose-dependent when evaluated with an appropriate trend test
•Any of the results are outside the distribution of the historical vehicle control data

b.A test chemical is considered to be clearly negative if, in all experimental conditions examined:

•None of the test concentrations exhibits a statistically significant increase in number of aberrations compared with the concurrent vehicle control
•There is no concentration-related increase when evaluated with an appropriate trend test
•All results are inside the distribution of the historical vehicle control data

c.The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed.
Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

TABLE 1.         Determination of pH of Test Medium

Treatment (mg/mL)	pH at the beginning of exposure to treatment		pH at the end of exposure to treatment
	With S9	Without S9	With S9	Without S9
DMSO	7.09	7.22	7.01	7.07
8	7.11	7.31	7.00	7.07
16	7.12	7.26	6.99	7.12
32	7.14	7.26	7.02	7.25
64	7.14	7.27	7.02	7.21
128	7.13	7.28	7.00	7.14
256	7.14	7.28	7.04	7.14
512	7.15	7.27	7.03	7.18
1024	7.14	7.30	7.02	7.20
2000	7.14	7.33	7.04	7.17

TABLE 2.         Determination of Osmolality of Test Medium

Treatment (mg/mL)	Osmolality at the beginning of exposure to treatment (OSMOL/kg)		Osmolality at the end of exposure to treatment (OSMOL/kg)
	With S9	Without S9	With S9	Without S9
DMSO	0.467	0.466	0.464	0.469
64	-	-	0.455	0.456
128	-	-	0.452	0.464
2000	0.437	0.437	0.430	0.433

TABLE 3.         Results of Preliminary Cytotoxicity Test

Treatment (mg/mL)	3-hour exposure with metabolic activation				3-hour exposure without metabolic activation
	Cloning Efficiency (CE)	Cells at end of treatment (1x10^5/mL)	Adjusted Cloning Efficiency	Relative Survival (%RS)*	Cloning Efficiency (CE)	Cells at end of treatment (1x10^5/mL)	Adjusted Cloning Efficiency	Relative Survival (%RS)
DMSO	0.96	12.4	1.39	100	0.95	13.35	1.48	100
8	0.89	11.80	1.22	88	0.86	12.45	1.25	84
16	0.84	11.45	1.12	81	0.78	12.15	1.11	75
32	0.76	9.90	0.88	63	0.75	10.75	0.94	64
64	0.67	9.20	0.72	52	0.66	10.05	0.77	52
128	0.60	9.50	0.66	47	0.55	10.00	0.64	43
256	0.52	9.05	0.55	40	0.58	9.45	0.64	43
512	0.49	8.80	0.50	36	0.52	8.35	0.51	34
1024	0.43	8.05	0.40	29	0.37	7.90	0.34	23
2000	0.34	7.45	0.30	22	0.32	7.80	0.29	20

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
DMSO	198	195	199	0.99	1.03	100
	199	196	198
250	99	109	106	0.53	0.52	50
	108	101	110
500	88	81	86	0.42	0.40	39
	79	84	82
1000	64	70	72	0.35	0.29	28
	71	74	69
2000	58	54	56	0.29	0.22	21
	60	58	59
3-MCA	130	123	126	0.64	0.46	45
	121	135	127

CE: Cloning EfficiencyACE: Adjusted CE                 RS: Relative Survival   

TABLE 5.         Parallel Cytotoxicity Test Results from Experiment 2

Treatment µg/mL	No. of Colonies /Flask			CE	ACE	RS %
	1	2	3
DMSO	194	193	196	0.96	0.95	100
	194	186	191
250	104	109	100	0.51	0.48	51
	106	100	98
500	98	91	89	0.45	0.42	44
	88	90	85
1000	74	74	78	0.37	0.32	34
	76	69	72
2000	68	70	62	0.32	0.22	23
	59	65	64

TABLE 6.         Summary Results of the Gene Mutation Assay in the Presence of Metabolic Activation (Experiment 1)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE
DMSO	3	1	2	1	2	18	0.000009	186	192	190	0.96	9.38
	1	2	2	2	2			194	191	193
250	2	0	1	3	1	13	0.000007	176	184	186	0.89	7.86
	1	1	2	1	1			169	179	177
500	0	2	1	1	1	12	0.000006	158	149	162	0.79	7.59
	1	3	2	1	0			163	159	160
1000	2	1	1	0	1	11	0.000006	148	131	141	0.71	8.45
	1	1	2	1	1			142	147	140
2000	2	1	0	1	1	11	0.000006	126	111	127	0.60	10.00
	3	1	0	1	1			123	110	119
3-MCA	30	33	31	35	33	322	0.000161	138	146	139	0.72	223.61
	32	31	31	34	32			154	139	145

Positive Control: 8 µg/mL 3-MCA             Vehicle Control: DMSO               CE: Cloning Efficiency                MF: Mutant Frequency

TABLE 7.         Summary Results of the Gene Mutation Assay in the Absence of Metabolic Activation (Experiment 2)

Treatment µg/mL	Mutation Assay Flasks						Cloning Efficiency of Mutant Colonies	Cloning Efficiency Flasks				6-TG Mutants per 10^6Clonable Cells (MF)
	TG Colonies/Flask							No. of Colonies/Flask
	1	2	3	4	5	Total		1	2	3	CE*
DMSO	1	2	2	2	3	21	0.000011	186	188	184	0.93	11.83
	2	1	3	4	1			190	185	187
250	2	1	1	2	3	16	0.000008	169	174	177	0.86	9.30
	1	3	1	1	1			171	170	173
500	2	1	1	2	1	14	0.000007	149	158	153	0.76	9.21
	0	2	2	1	2			138	149	159
1000	2	3	0	1	2	14	0.000007	132	136	123	0.64	10.94
	2	1	1	1	1			128	129	125
2000	1	1	2	1	1	13	0.000007	111	123	119	0.60	11.67
	1	2	1	1	2			126	120	117

Vehicle Control: DMSO              CE: Cloning Efficiency        MF: Mutant Frequency                                        

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test item, Fat Blue B01 does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Executive summary:

The genotoxic potential of the test item to induce gene mutation in mammalian cells was evaluated using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The test item formed a free flowing suspension in Dimethyl sulphoxide (DMSO) at 200 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 22 and 20% at the top concentration of 2000 µg/mL, in the presence and absence of metabolic activation, respectively. The test item precipitated in the test medium at and above 128 µg/mL, but did not show any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of
2000 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 250, 500, 1000 and 2000 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) were also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. The positive control in experiment 1 produced a statistically significant increase in the frequencies of mutants, under identical conditions.

 

The results of the forward gene mutation test at the hprt-locus indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

The test item did induce gene mutations by frameshifts in the genome of strain TA 98 with S9 mix. However, no mutagenic effects were detected in the hprt-locus in CHO cells.

Justification for selection of genetic toxicity endpoint
more recent studies and according to GLP and guideline

Justification for classification or non-classification

not classified;

positive findings in an Ames test are not sufficient for classification. Additionally the findings could not be confirmed in a genemutation assay in CHO cells at the hprt locus.