Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Sep 2016 - 08 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

other: HPRT

Test material

Test material information

Constituent 1

Reference substance name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

EC Number:

255-527-0

EC Name:

Sodium bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)

Cas Number:

41741-86-0

Molecular formula:

C34 H24 Cr N8 O6 .Na

IUPAC Name:

Sodium;chromium(3+);2-[(3-methyl-5-oxido-1-phenylpyrazol-4-yl)diazenyl]benzoate

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: doubling time of about 12 - 16 hours
Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS). Aall incubations were performed at 37°C with a relative humidity of ≥ 90% in a
5% (v/v) CO2 atmosphere.
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Based on the data and the observations from the pretest and taking into account the current guidelines, the following doses were selected in this study:
1st Experiment:
without S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL
with S9 mix: 0; 0.16; 0.31; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00 μg/mL
2nd Experiment:
without S9 mix: 0; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL
with S9 mix: 0; 0.94; 1.88; 3.75; 7.50; 15.00; 30.00 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, dimethyl sulfoxide (DMSO) was selected as vehicle, which has been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data are available. The final concentration of the vehicle DMSO in culture medium was 1% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 – 9 days
- Selection time (if incubation with a selection agent): 6 – 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 16 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency 1 (CE1; survival)
For the determination of the influence of the test substance after the exposure period, about 200 cells per concentration will be reserved from the treated cells and will be seeded in two 60 mm petri dishes and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency 2 (CE2; viability)
For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS. In all cases, after seeding the flasks or petri dishes were incubated for 6 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH values were not influenced by test substance treatment
- Effects of osmolality: Osmolality was not influenced by test substance treatment
- Precipitation: In this study, without metabolic activation test substance precipitation in culture medium was observed from about 40.0 μg/mL onward in both main experiments. In the presence of S9 mix no precipitation in culture medium at the end of exposure was found up to the highest applied concentration.

RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 2000 μg/mL test material was used as top concentration both with and without S9 mix at 4 hour exposure time. The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses. In the pretest the pH value was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, precipitation of the test substance in the vehicle DMSO was not observed in the stock solution (Test group: 2000.0 μg/mL). In culture medium, test substance precipitation occurred by the end of treatment at concentrations of 1000.0 μg/mL and above in the absence of S9 mix and at concentrations of 250.0 μg/mL and above in the presence of S9 mix. After 4 hours treatment in the absence of S9 mix, cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival at 31.3 μg/mL and above. Additionally, in the presence of S9 mix, clearly reduced relative cloning efficiency was observed from the lowest applied concentration of 7.8 μg/mL onward.

HISTORICAL CONTROL DATA
The mutation frequencies of the vehicle control groups were within the historical negative control data range (95% control limit). In addition, the increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were observed in both experiments in the presence of S9 mix, at the highest applied concentrations. In detail, without S9 mix, there was a clear decrease in the number of colonies after an exposure period of 4 hours at 40.0 μg/mL (CE1 relative: 7.3%) in the 1st Experiment and at 60.0 μg/mL (CE1 relative: 1.1%) in the 2nd Experiment. In addition, with S9 mix, there was a strong decrease in the number of colonies at 20.0 μg/mL (CE1 relative: 3.2%) in the 1st Experiment and at 30.0 μg/mL (CE1 relative: 0.3%) in the 2nd Experiment. Although the substance was diluted using steps of 2-fold, cloning efficiency values in the range of 10 to 20% were not obtained in both experiments, due to a steep increase of test substance cytotoxicity.

Any other information on results incl. tables

No biologically relevant increase in the number of mutant colonies was observed with or without S9 mix. In detail, in the absence of S9 mix, in the 1st Experiment the values for the corrected mutation frequencies (MFcorr.: 0.49 – 4.40 per 10⁶ cells) were close to the concurrent vehicle control value (MFcorr.: 2.38 per 10⁶ cells) and clearly within the range of the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 7.19 per 10⁶ cells). In the 2nd Experiment at the two lowest applied test substance concentrations (1.88 and 3.75 μg/mL) unexpectedly high mutant frequencies (MFcorr.: 12.13 and 18.93 per 10⁶ cells, respectively) were obtained being statistically significant increased compared to the respective vehicle control value (MFcorr.: 5.00 per 10⁶ cells) and clearly exceeded the historical negative control data range (MFcorr.: 0.00 – 9.93 per 10⁶ cells). Due to the almost inverse dose-relation in this case and a comparable observation in a different study performed in the same time period in our laboratory (internal data) it has to be speculated that a technical error (i.e. improper test culture material) occurred in these test groups. Thus, these observations were considered biologically irrelevant. In this experimental part at the lower end of the test substance concentrations tested (7.50 to 30.0 μg/mL) the values for the corrected mutation frequencies (MFcorr.: 4.35 – 7.94 per 10⁶ cells) were close to the concurrent vehicle control value (MFcorr.: 5.00 per 10⁶ cells) and below or close to the range of the 95% control limit of the historical negative control data (MFcorr.: 0.00 – 7.19 per 10⁶ cells). In addition, in both experiments in the absence of S9 mix no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment. In the presence of S9 mix, in the 1st Experiment the values for the corrected mutation frequencies (MFcorr.: 0.00 – 3.46 per 10⁶ cells) were close to the concurrent vehicle control value (MFcorr.: 3.45 per 10⁶ cells) and clearly within the range of the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 7.19 per 106 cells). In the 2nd Experiment the values for the corrected mutation frequencies (MFcorr.: 2.98 – 8.06 per 10⁶ cells) were close to the concurrent vehicle control value (MFcorr.: 3.60 per 10⁶ cells) and close to the range of the 95% control limit of our historical negative control data (MFcorr.: 0.00 – 7.19 per 10⁶ cells). In addition, in both experiments in the presence of S9 mix no statistically significant dose-related increase in the mutant frequency was found in cells after 4 hours of treatment.

Summary of Results

						Cytotoxicity***
Exp	Exposure period [h]	Test groups [μg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6 cells]	CE1 [%]	CE2 [%]
1	4	Vehicle control^1	-	n.d.	2.38	100	100
		0.63	-	-	n.c.^1	88.4	n.c.^1
		1.25	-	-	3.49	91.6	102.4
		2.5	-	-	4.40	77.8	99.2
		5	-	-	1.23	104.6	96.8
		10	-	-	1.30	84.1	91.3
		20	-	-	0.40	75.7	98.4
		40	-	+	n.c.^2	7.3	n.c.^2
		Positive control^2	-	n.d.	138.78^S	55.1	77.8
2	4	Vehicle control^1	-	n.d.	5.00	100	100
		1.88	-	-	12.13^S	63.4	90.7
		3.75	-	-	18.93^S	71.1	105.7
		7.5	-	-	7.94	62.1	92.3
		15	-	-	4.35	71.1	99.7
		30	-	-	5.56	63.6	96
		60	-	+	n.c.^2	1.1	n.c.^2
		Positive control^2	-	n.d.	259.63^S	53.2	72.7
1	4	Vehicle control^1	+	n.d.	3.45	100	100
		0.16	+	-	n.c.^1	104.1	n.c.^1
		0.31	+	-	n.c.^1	88.9	n.c.^1
		0.63	+	-	3.46	77	88.5
		1.25	+	-	2.97	71.1	90.4
		2.5	+	-	0.00	73.2	78.2
		5	+	-	1.83	60.3	83.9
		10	+	-	0.00	46.9	79.3
		20	+	-	n.c.^2	3.2	n.c.^2
		Positive control^3	+	n.d.	114.21^S	63.3	72.8
2	4	Vehicle control^1	+	n.d.	3.6	100	100
		0.94	+	-	4.1	91.2	80.5
		1.88	+	-	8.06	82.8	100.6
		3.75	+	-	4.74	126.3	69.7
		7.5	+	-	3.96	72.4	83.5
		15	+	-	2.98	31.7	70.6
		30	+	-	n.c.^2	0.3	n.c.^2
		Positive control^3	+	n.d.	171.59^S	65.8	52.9

*        Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE2 value

***  Cloning efficiency related to the respective vehicle control

^S       Mutant frequency statistically significant higher than corresponding control values

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations is required

n.c.² Culture was not continued due to strong cytotoxicity

n.d. Not determined

¹ DMSO 1% (v/v)

² EMS 400 μg/mL

³ DMBA 1.25 μg/mL

Applicant's summary and conclusion

Conclusions:

The test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. Test groups printed in bold type were evaluated for gene mutations:

1st Experiment

without S9 mix: 0; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00; 40.00 μg/mL

with S9 mix: 0; 0.16; 0.31; 0.63; 1.25; 2.50; 5.00; 10.00; 20.00 μg/mL

2nd Experiment

without S9 mix: 0; 1.88; 3.75; 7.50; 15.00; 30.00; 60.00 μg/mL

with S9 mix: 0; 0.94; 1.88; 3.75; 7.50; 15.00; 30.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in the 1st and 2nd Experiment, the highest concentrations tested for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. In the 2nd Experiment in the absence of S9 mix the two lowest applied test substance concentrations led to statistically significant increased mutant frequencies. These findings occurred due to a technical error and, thus, haave to be regarded as biologicaly irrelevant. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.