Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (Ames) no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor 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.

The results of the forward gene mutation test at the hprt locus with the test item indicated that the test item was non-mutagenic.

In an in vitro chromosomal aberration study a total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations. There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. The study indicated that the test item was not clastogenic.

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:

2016 -04-19 till 2016-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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 in experiment I and non-induced hamster liver S9 in experiment II

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [deionised water
- Justification for choice of solvent/vehicle: best suitable solvent

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

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

Details on test system and experimental conditions:

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


DURATION
- Preincubation period: 30 min
- 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:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in strain TA 1535, TA 1537 and TA 98

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: yes
- Precipitation:No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.
- Other confounding effects:
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):
Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 1535 / / / 5000
TA 1537 / 5000 5000 /
TA 98 / 5000 / /
TA 100 / / / /
WP2 uvrA / / / /

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Summary Tabellen

Table1            Summary of Experiment I

Study Name: 1763502	Study Code: Envigo 1763502
Experiment: 1763502 VV Plate	Date Plated: 19.04.2016
Assay Conditions:	Date Counted: 26.04.2016

 

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	Deion. water		11 ± 4	9 ± 2	29 ± 4	186 ± 16	54 ± 6
	Untreated		12 ± 3	9 ± 4	32 ± 6	157 ± 14	52 ± 11
	Sanodal-	3 µg	9 ± 1	10 ± 3	34 ± 4	172 ± 4	49 ± 11
	Schwarz 2LW	10 µg	15 ± 5	7 ± 2	26 ± 5	172 ± 17	51 ± 7
		33 µg	11 ± 1	9 ± 1	27 ± 6	157 ± 6	41 ± 4
		100 µg	13 ± 6	11 ± 5	28 ± 6	166 ± 28	61 ± 6
		333 µg	12 ± 3	6 ± 1	29 ± 3	168 ± 12	48 ± 5
		1000 µg	10 ± 2^D M	8 ± 4^D M	26 ± 7^D M	149 ± 4^D M	43 ± 6^D M
		2500 µg	10 ± 3^D M	8 ± 3^D M	19 ± 5^D M	146 ± 7^D M	36 ± 4^D M
		5000 µg	8 ± 2^D M	7 ± 1^D M	15 ± 1^D M	135 ± 5^D M	36 ± 3^D M
	NaN3	10 µg	1382 ± 52			2415 ± 69
	4-NOPD	10 µg			403 ± 23
	4-NOPD	50 µg		75 ± 7
	MMS	2.0 µL					1075 ± 78
With Activation	Deion. water		10 ± 2	9 ± 3	40 ± 6	170 ± 20	63 ± 5
	Untreated		13 ± 4	10 ± 5	45 ± 7	165 ± 20	61 ± 14
	Sanodal-	3 µg	10 ± 1	11 ± 4	44 ± 6	167 ± 8	62 ± 8
	Schwarz 2LW	10 µg	12 ± 5	9 ± 3	48 ± 6	172 ± 18	53 ± 10
		33 µg	8 ± 2	11 ± 2	37 ± 5	172 ± 16	52 ± 4
		100 µg	10 ± 0	8 ± 3	40 ± 10	173 ± 21	69 ± 11
		333 µg	9 ± 5	11 ± 4	34 ± 2	174 ± 25	54 ± 13
		1000 µg	10 ± 1^D M	8 ± 2^D M	27 ± 4^D M	135 ± 6^D M	42 ± 1^D M
		2500 µg	7 ± 3^D M	5 ± 2^D M	23 ± 6^D M	115 ± 8^D M	40 ± 2^D M
		5000 µg	5 ± 2^D M	3 ± 1^D M	17 ± 2^D M	106 ± 7^D M	30 ± 4^D M
	2-AA	2.5 µg	415 ± 21	219 ± 43	6586 ± 361	4872 ± 201
	2-AA	10.0 µg					414 ± 15

 

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	Densely coloured plate Manual count

 

 

Table2            Summary of Experiment II

Study Name: 1763502	Study Code: Envigo 1763502
Experiment: 1763502 HV2 Pre	Date Plated: 12.05.2016
Assay Conditions:	Date Counted: 18.05.2016

 

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	Deion. water		10 ± 1	13 ± 3	29 ± 2	146 ± 22	51 ± 9
	Untreated		12 ± 7	12 ± 2	32 ± 8	169 ± 11	52 ± 7
	Sanodal-	3 µg	11 ± 1	11 ± 1	26 ± 9	161 ± 8	41 ± 1
	Schwarz 2LW	10 µg	12 ± 6	10 ± 2	25 ± 6	167 ± 13	42 ± 2
		33 µg	13 ± 3	12 ± 6	28 ± 2	148 ± 22	50 ± 5
		100 µg	13 ± 3	11 ± 3	27 ± 12	154 ± 8	37 ± 3
		333 µg	14 ± 4^P	10 ± 1^P	32 ± 9^P	167 ± 7^P	32 ± 8^P
		1000 µg	7 ± 3^P M	8 ± 2^P M	16 ± 2^P M	159 ± 6^P M	29 ± 4^M P
		2500 µg	7 ± 2^P M	8 ± 2^P M	14 ± 2^P M	157 ± 14^P M	28 ± 7^M P
		5000 µg	7 ± 2^P D M	4 ± 1^P D M	15 ± 3^P D M	143 ± 6^P D M	28 ± 9^M P D
	NaN3	10 µg	1140 ± 35			1849 ± 49
	4-NOPD	10 µg			447 ± 25
	4-NOPD	50 µg		72 ± 12
	MMS	2 µL					879 ± 65
With Activation	Deion. water		18 ± 4	25 ± 3	50 ± 2	140 ± 32	46 ± 4
	Untreated		18 ± 4	23 ± 9	57 ± 10	123 ± 19	50 ± 11
	Sanodal-	3 µg	22 ± 0	29 ± 2	57 ± 3	147 ± 14	51 ± 3
	Schwarz 2LW	10 µg	20 ± 4	30 ± 6	55 ± 13	150 ± 11	48 ± 3
		33 µg	22 ± 6	31 ± 10	55 ± 6	177 ± 13	42 ± 4
		100 µg	13 ± 1	30 ± 6	49 ± 8	167 ± 32	45 ± 11
		333 µg	16 ± 5^P	29 ± 4^P	51 ± 4^P	143 ± 7^P	49 ± 4^P
		1000 µg	13 ± 3^P M	30 ± 8^P M	36 ± 3^P M	139 ± 12^P M	31 ± 5^P M
		2500 µg	11 ± 3^P M	21 ± 3^P M	29 ± 4^P M	132 ± 11^P M	33 ± 8^P M
		5000 µg	8 ± 2^P D M	18 ± 3^P D M	23 ± 3^P D M	123 ± 2^P D M	26 ± 9^P D M
	2-AA	2.5 µg				963 ± 99
	2-AA	2.5 µg	329 ± 27	115 ± 14
	2-AA	10 µg					1043 ± 60
	Congo red	500 µg			328 ± 10

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P M D	Precipitate Manual count Densely coloured plate

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

 

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

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

No precipitation occurred in the overlay agar in the test tubes at any concentration. In Experiment I the minimal agar plates were densely colored after treatment with the test items at concentrations ranging from 1000 to 5000 µg/plate. In Experiment II only after treatment with 5000 µg/plate densely colored plates were recognized.

Furthermore in Experiment II precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate.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.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	5000
TA 1537	/	5000	5000	/
TA 98	/	5000	/	/
TA 100	/	/	/	/
WP2 uvrA	/	/	/	/

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sanodal-Schwarz 2LW at any concentration level, neither in the presence nor 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. They showed a distinct in­crease in induced revertant colonies.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Cell cultures were exposed to the test item for 3 hours both in the presence and absence of exogenous metabolic activation and for 21 hours in the absence of metabolic activation. Following exposure of cell cultures to the test item, they were treated with Colchicine to arrest the cells in a metaphase-like stage of mitosis (c-metaphase). Cells were then harvested and chromosome preparations made. Preparations were stained and metaphases were analyzed for chromosomal aberrations.

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:

Experiment No. Test Concentrations
Experiments 1 & 2: a) 12 b) 60 and c) 300 µg/mL (factor of 5)
Experiment 3: a) 6 b) 32 and c) 160 µg/mL (factor of 5)

Vehicle / solvent:

sterile water was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

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 results will be considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required to clarify such results.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

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.         Preliminary Cytotoxicity Test - 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
SW	7.02	7.02	7.05	7.16
5	7.07	7.13	7.08	7.11
10	7.06	7.17	7.01	7.19
20	7.08	7.18	7.07	7.19
40	7.10	7.19	7.02	7.15
80	7.10	7.18	7.06	7.17
160	7.08	7.19	7.02	7.20
320	7.10	7.20	7.02	7.15
500	7.08	7.17	7.09	7.15

Note: No precipitation was observed both in the presence and absence of metabolic activation at any of the tested concentrations at 3 hours post treatment.

TABLE 2.    Preliminary Cytotoxicity Test - 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
SW	0.317	0.311	0.329	0.320
500	0.319	0.311	0.328	0.321

TABLE 3.    Results of Preliminary Cytotoxicity Test

 

Treatment (mg/mL)	Presence of metabolic activation (3-hour exposure)			Absence of metabolic activation (3-hour exposure)			Absence of metabolic activation (21-hour exposure)
	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)	Final – initial cell count (1x10^6/flask)	Cell growth index RICC (%)	Cell growth inhibition (%)
SW	1.425	100	0	1.175	100	0	1.1375	100	0
5	1.175	82	18	1.025	87	13	0.9125	80	20
10	1.0125	71	29	0.925	79	21	0.775	68	32
20	0.925	65	35	0.8875	76	24	0.6875	60	40
40	0.8625	61	39	0.7875	67	33	0.650	57	43
80	0.80	56	44	0.675	57	43	0.55	48	52
160	0.75	53	47	0.6125	52	48	0.525	46	54
320	0.60	42	58	0.4875	41	59	0.075	7	93
500	0.425	30	70	0.30	26	74	0.0875	8	92

Note: Initial cell count (No. of cells at the beginning of treatment): 1.45 x 10⁶cells/flask .

TABLE 4.    Summary Results of Chromosomal Aberration Assay - Experiment 1

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
SW	300	0	0	0	0	0	0	0	0	0	0
12	300	2 (0.7)	0	0	0	0	0	0	2 (0.7)	0	17
60	300	0	1 (0.3)	0	0	0	0	0	1 (0.3)	0	39
300	300	1 (0.3)	2 (0.7)	0	0	0	0	0	3 (1.0)	0	52
CPA 55	300	0	0	32 (10.7)	71 (23.7)	24 (8.0)	24 (8.0)	0	141 (47.0)	+ 141 (47.0)	21

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

CPA: Cyclophosphamide monohydrate            +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells.

TABLE 5.    Summary Results of Chromosomal Aberration Assay - Experiment 2

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
SW	300	0	2 (0.7)	0	0	0	0	0	2 (0.7)	0	0
12	300	0	0	0	0	0	0	0	0	0	19
60	300	0	0	0	0	0	0	0	0	0	43
300	300	0	0	0	0	0	0	0	0	0	58

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type                  RC: Ring chromosome 

Note: There were no incidences of polyploidy and endoreduplicated cells

TABLE 6.    Summary Results of Chromosomal Aberration Assay - Experiment 3

Treatment (µg/mL)	No. of metaphases scored	No. (%) of metaphases with aberrations^@							Total No.(%) of aberrant metaphases^*		Cell Growth Inhibition (%)
		Gaps		Breaks		Exchanges			Including Gaps	Excluding Gaps
		Cs	Ct	Cs	Ct	Cs	Ct	RC
SW	300	1 (0.3)	1 (0.3)	0	2 (0.7)	0	0	0	4 (1.3)	2 (0.7)	0
6	300	0	2 (0.7)	0	1 (0.3)	0	0	0	3 (1.0)	1 (0.3)	21
32	300	1 (0.3)	1 (0.3)	0	2 (0.7)	0	0	0	4 (1.3)	2 (0.7)	43
160	300	1 (0.3)	0	0	0	0	0	0	1 (0.3)	0	52
EMS600	300	8 (2.7)	9 (3.0)	41 (13.7)	75 (25.0)	52 (17.3)	28 (9.3)	0	151 (50.3)	+ 151 (50.3)	33

*: Metaphase plate with one or more than one aberrations considered as one metaphase plate with aberrations

@: values are the sum of two replicates and values in parenthesis represent %   

Cs: Chromosome type             Ct: Chromatid type      RC: Ring chromosome    

EMS: Ethyl methanesulfonate   +: Significantly higher than control (p <0.05) by Fischer exact test

Note: There were no incidences of polyploidy and endoreduplicated cells

Conclusions:

Interpretation of results (migrated information):
negative

The test item was not clastogenic in CHO-K1 cells at the tested concentration (up to 300 µg/mL) and under the conditions of testing employed

Executive summary:

The clastogenic potential of the test item to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in sterile water (SW) at 50 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, CHO-K1 cells exposed to the test item, exhibited required level of cytotoxicity as RICC between 160 and 500 µg/mL, compared to the SW control, both in the presence and absence of metabolic activation with 3-hour exposure. In the absence of metabolic activation with 21-hour exposure, required level of cytotoxicity was observed between 40 and 320 µg/mL compared to the SW. Based on these observations, a maximum of 300 µg/mL in experiments 1 and 2 and a maximum of 160 µg/mL in experiment 3 of the chromosomal aberration assay was tested.

 

In the definitive chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for 3 hours at concentrations of 12, 60 and 300mg/mL in experiments 1 and 2 (presence and in the absence of metabolic activation, respectively). Similarly, in experiment 3, CHO-K1 cells were exposed to the test item in duplicate for 21 hours at concentrations of 6, 32, and 160 µg/mL in the absence of metabolic activation. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the respective highest concentrations tested, the reduction in cell growth as RICC was 52, 58 and 52% in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.

There was no evidence of induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

 

The study indicated that the test item was not clastogenic at the concentrations tested and under the conditions of testing.

Reference 3

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

according to OECD and GLP

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) 62.5 B) 125 C) 250 and D) 500 µg/mL (factor of 2)

Vehicle / solvent:

sterile water was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

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).

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
SW	7.12	7.15	7.08	7.16
5	7.14	7.16	7.10	7.19
10	7.16	7.18	7.10	7.22
20	7.16	7.18	7.11	7.24
40	7.17	7.16	7.10	7.23
80	7.18	7.20	7.12	7.24
160	7.18	7.23	7.10	7.24
320	7.19	7.25	7.09	7.23
500	7.20	7.25	7.08	7.24

Note: There was no precipitation of the test item at any of tested concentrations either in the presence or absence of metabolic activation at 3 hours post treatment.

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
SW	0.317	0.311	0.329	0.320
500	0.319	0.311	0.328	0.321

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)*
SW	0.97	12.90	1.44	100	0.97	12.45	1.39	100
5	0.88	12.00	1.21	84	0.94	11.80	1.27	91
10	0.82	10.95	1.03	72	0.86	11.35	1.12	81
20	0.80	10.35	0.95	66	0.79	10.50	0.95	68
40	0.74	9.15	0.78	54	0.76	9.30	0.81	58
80	0.65	7.05	0.53	37	0.71	8.50	0.69	50
160	0.62	6.90	0.49	34	0.66	7.20	0.55	40
320	0.57	5.15	0.34	24	0.58	4.90	0.33	24
500	0.47	4.95	0.27	19	0.48	3.80	0.21	15

TABLE 4.         Parallel Cytotoxicity Test Results from Experiment 1

Treatment µg/mL	No. of Colonies /Flask			CE*	ACE	RS %
	1	2	3
SW	191	189	195	0.968	1.41	100
	197	193	197
62.5	137	141	135	0.707	0.72	51
	145	147	143
125	138	131	136	0.670	0.55	39
	129	132	138
250	126	127	130	0.633	0.46	33
	136	121	119
500	111	118	99	0.547	0.28	20
	106	109	113
3-MCA	136	148	131	0.716	0.72	51
	152	143	149

CE: Cloning Efficiency ; ACE: 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
SW	197	195	196	0.974	1.49	100
	191	197	193
62.5	153	160	149	0.763	0.77	52
	147	156	151
125	141	138	135	0.69	0.62	42
	143	140	131
250	121	127	120	0.616	0.50	34
	119	129	123
500	110	113	97	0.533	0.24	16
	107	101	112

CE: Cloning EfficiencyACE: Adjusted CE                 RS: Relative Survival   

* calculated from the mean values of the replicates of each group and rounded off to two decimal places

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*
SW	1	1	3	1	1	16	0.000008	188	189	193	0.95	8.42
	2	3	1	1	2			191	194	190
62.5	0	1	2	1	1	12	0.000006	183	172	176	0.89	6.74
	1	2	1	1	2			181	179	174
125	3	0	2	1	1	12	0.000006	158	162	171	0.83	7.23
	1	1	1	1	1			172	170	164
250	1	1	1	1	2	12	0.000006	157	149	158	0.77	7.79
	0	2	2	1	1			146	151	159
500	2	2	1	0	0	11	0.0000055	141	136	133	0.67	8.21
	2	1	1	1	1			133	130	135
3-MCA	36	34	38	29	26	315	0.0001575	148	150	141	0.75	210^+
	28	29	31	34	30			153	157	146

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*
SW	2	2	2	3	1	22	0.000011	191	196	187	0.95	11.58
	2	3	2	2	3			189	190	192
62.5	1	1	2	2	2	17	0.0000085	178	180	191	0.92	9.24
	2	3	1	1	2			184	185	183
125	0	3	1	1	2	15	0.0000075	163	167	160	0.83	9.04
	4	2	0	1	1			169	165	166
250	1	1	1	1	2	14	0.000007	148	153	154	0.76	9.21
	2	1	2	2	1			150	157	152
500	1	1	2	2	1	14	0.000007	143	149	137	0.71	9.86
	2	3	1	1	0			135	141	145

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

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that the test item 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 assay. The gene mutation assay 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 sterile water (SW) at 50 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the Relative Survival was 19 and 15 % at the 500 µg/mL, in the presence and absence of metabolic activation, respectively. The test item neither precipitated in the test medium at the highest concentration nor showed any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of
500 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation assay, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 62.5, 125, 250 and 500 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 (SW) 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 with the test item indicated that the test item was non-mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No relevant findings observed.

Justification for classification or non-classification

No classification

All studies (Ames, HPRT, Chromosomal Aberration) did not afford any indication of mutagenicity.