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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03-07-2014 - 03-03-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cells forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: flakes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: N10415
- Expiration date of the lot/batch: 30-06-2015
- Purity test date: 07-11-2013

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator, dry storage (from 14 Mar 2014 onwards),
light protected conditions (from 15 Jul 2014 onwards)
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 30 Jun 2015 as indicated by the sponsor and the sponsor holds this responsibility.
The stability of the test substance under the below described storage conditions without light protection was confirmed by reanalyses.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is light sensitive and easily decomposes under light exposure as indicated by the sponsor. This study was not performed under light protection conditions. Therefore, it was not clear if the adverse effects observed in the experiments were induced by the test substance or by decomposition products of the test substance after light exposure. Thus, an additional investigation on test substance stability under the chosen light conditions of the experiments in the laboratory was performed.
The probes were incubated under three different conditions to examine the light- and/or temperature-dependent decomposition of the test substance over a period of 4 hours. The conditions of the common workflow in the laboratory – standard light at clean bench, room temperature – were compared with light protected storage at room temperature (about 20°C) or incubator temperature (37°C). The analyses were carried out following Control procedure No. 14/0001_02-01.
A test substance preparation with a final concentration of 1 mg/mL was prepared in supplemented culture medium including S9 mix (see Paragraph 3.5 and/or Table 6). The preparation was aliquoted in 25 cm2 plastic flasks (5 mL each) similar to the standard procedure used in the laboratory. Then, the probes were incubated for the intended time on the clean bench under standard light or in a dark cupboard at room temperature or in an incubator at 37°C. After reaching the intended time point (0; 30; 60; 120; 240 min) the probes were placed light protected on ice and were transferred directly to the analytical laboratory.
The test substance concentrations were determined immediately after sampling and shipping.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The substance was dissolved in culture medium (Ham’s F12). To achieve a solution of the test substance in the vehicle, the test substance preparation was pipetted thoroughly.
The further concentrations were diluted from the stock solution according to the planned doses.
All test substance solutions were prepared immediately before administration.

Method

Target gene:
HPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in liquid nitrogen using
7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity
testing was checked for mycoplasma contamination.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Ham's F12 medium containing stable glutamine and hypoxanthine (Biochrom; Cat. No.
FG 0815) supplemented with 10% (v/v) fetal calf serum (FCS). With
- 1% (v/v) penicillin/streptomycin (stock solution: 10 000 IU / 10 000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Cells were grown with 5% (v/v) CO2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 fraction
Test concentrations with justification for top dose:
1st Experiment, without S9 mix, 4-hour exposure
62.5; 125; 250; 500; 1000; 2000 µg/mL
1st Experiment, with S9 mix, 4-hour exposure
125; 250; 500; 1000; 2000; 3000 µg/mL
2nd Experiment, without S9 mix, 4-hour exposure
78.1; 156.3; 312.5; 625; 1250; 2500 µg/mL
2nd Experiment, with S9 mix, 4-hour exposure
78.1; 156.3; 312.5; 625; 1250; 2500; 3300 µg/mL
3rd Experiment, with S9 mix, 4-hour exposure
1000; 1500; 2000; 2500; 3000; 3300 µg/mL

Justification:
Results of Pre-Test
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 1 650 μg/mL and
above. In the presence of S9 mix, a clearly reduced relative cloning efficiency was observed
at the highest applied concentration of 3 300 μg/mL
Vehicle / solvent:
Due to the good solubility of the test substance in water, culture medium (Ham's F12) was
selected as vehicle.
Controls
Untreated negative controls:
yes
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;
- Cell density at seeding (if applicable): 10E06

- Exposure duration: 4h
- 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):13-16 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS:
Duplicate cultures
six replicates per culture prepared for selection

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
- Method: cloning efficiency


Rationale for test conditions:
standard conditions as layed down in Guideline
Evaluation criteria:
Mutant frequency; The number of colonies in every flask was counted and recorded. The sum of the mutant colony
counts within each test group was subsequently normalized per every 106 cells seeded.
Statistics:
An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
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: The pH was measured for the top concentration and for the vehicle controls with and without S9 mix each.
- Effects of osmolality: Osmolarity was measured in the top concentration and the negative controls with and without S9 mix each.
- Evaporation from medium: not relevant
- Water solubility: not relevant/see below
- Precipitation: Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.
- Definition of acceptable cells for analysis: not relevant
- Other confounding effects: Cell morphology
The test cultures of all test groups were examined microscopically for cell morphology at the end of the exposure period, which allows to draw conclusions about the cellular attachment.

RANGE-FINDING/SCREENING STUDIES:
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 1 650 μg/mL and above. In the presence of S9 mix, a clearly reduced relative cloning efficiency was observed at the highest applied concentration of 3 300 μg/mL


HISTORICAL CONTROL DATA
for positive and negative contorls, the results were within the range of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values or severe cell loss at 1st passage were observed in all experiments in the absence and presence of S9 mix, at least at the highest applied concentrations.

Any other information on results incl. tables

Table 1: Mutant frequency – 1st Experiment without S9 mix, 4-Hour exposure period

Testgroups[µg/mL]

 

 

Numberofcoloniesa

Mutantfrequency(per106 cells)

 

Uncorrected

Correctedb

 

Negativecontrol*

A

1

0

0

0

0

0

 

0.28

 

0.29

B

0

0

0

0

0

0

 

62.5

A

 

n.c.1

 

n.c.1

 

n.c.1

B

 

125.0

A

0

0

0

0

0

0

 

2.22

 

2.42

B

1

0

3

0

2

2

 

250.0

A

0

0

0

0

0

0

 

0.00

 

0.00

B

0

0

0

0

0

0

 

500.0

A

0

0

0

0

0

0

 

0.00

 

0.00

B

0

0

0

0

0

0

 

1000.0

A

0

0

0

0

0

0

 

4.44

 

5.79

B

0

2

4

4

2

4

 

2000.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

EMS300

A

15

15

20

19

19

23

 

63.61

 

75.62

B

15

21

25

21

18

18

 

Table 2: Mutant frequency – 1st Experiment with S9 mix, 4-Hour exposure period

Testgroups[µg/mL]

 

 

Numberofcoloniesa

Mutantfrequency(per106 cells)

 

Uncorrected

Correctedb

 

Negativecontrol*

A

1

0

1

0

0

1

 

0.83

 

0.85

B

0

0

0

0

0

0

 

125.0

A

0

2

0

0

1

0

 

1.67

 

1.88

B

1

2

0

0

0

0

 

250.0

A

0

0

0

0

0

0

 

0.00

 

0.00

B

0

0

0

0

0

0

 

500.0

A

0

0

1

1

1

0

 

2.22

 

2.26

B

1

0

0

2

2

0

 

1000.0

A

1

2

2

2

0

1

 

3.06

 

3.18

B

1

0

0

0

1

1

 

2000.0

A

1

0

0

0

0

1

 

0.83

 

1.14

B

0

0

0

1

0

0

 

3300.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

DMBA1.25

A

40

30

33

30

37

33

 

118.33

 

158.25

B

38

39

30

37

47

32

 

Table 3: Mutant frequency –2nd Experiment without S9 mix, 4-Hour exposure period

Testgroups[µg/mL]

 

 

Numberofcoloniesa

Mutantfrequency(per106 cells)

 

Uncorrected

Correctedb

 

Negativecontrol*

A

0

1

0

0

2

1

 

2.50

 

2.77

B

0

1

1

2

0

1

 

78.1

A

4

3

2

1

3

2

 

4.44

 

5.34

B

1

0

0

0

0

0

 

156.3

A

0

0

0

0

1

1

 

1.67

 

1.94

B

1

1

1

1

0

0

 

312.5

A

0

1

0

0

1

0

 

0.56

 

0.66

B

0

0

0

0

0

0

 

625.0

A

1

0

0

1

0

0

 

0.56

 

0.68

B

0

0

0

0

0

0

 

1250.0

A

1

1

1

1

0

1

 

1.39

 

1.74

B

0

0

0

0

0

0

 

2500.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

EMS300

A

14

16

22

19

24

21

 

60.56

 

83.62

B

19

18

18

17

16

14

 

Table 4: Mutant frequency – 2nd Experiment with S9 mix, 4-Hour exposure period

Testgroups[µg/mL]

 

 

Numberofcoloniesa

Mutantfrequency(per106 cells)

 

Uncorrected

Correctedb

 

Negativecontrol*

A

1

0

1

1

0

1

 

1.39

 

1.80

B

1

0

0

0

0

0

 

156.3

A

 

n.c.1

 

n.c.1

 

n.c.1

B

 

312.5

A

0

1

0

0

2

0

 

1.94

 

2.56

B

2

0

0

1

1

0

 

625.0

A

0

1

1

1

2

0

 

3.06

 

3.97

B

1

0

2

0

2

1

 

1250.0

A

1

0

1

1

1

0

 

1.11

 

1.42

B

0

0

0

0

0

0

 

2500.0

A

5

9

8

7

3

4

 

10.28

 

14.85

B

0

0

1

0

0

0

 

3300.0

A

5

5

8

2

7

6

 

10.83

 

18.16

B

1

1

1

2

0

1

 

DMBA1.25

A

33

39

31

35

40

38

 

132.78

 

221.39

B

42

42

38

44

49

47

 

Table 5: Mutant frequency – 3rd Experiment with S9 mix, 4-Hour exposure period

Testgroups[µg/mL]

 

 

Numberofcoloniesa

Mutantfrequency(per106 cells)

 

Uncorrected

Correctedb

 

Negativecontrol*

A

0

1

0

2

1

1

 

6.11

 

8.20

B

4

6

4

0

0

3

 

1000.0

A

0

0

0

0

0

0

 

0.83

 

1.08

B

0

2

1

0

0

0

 

1500.0

A

0

0

1

0

0

0

 

1.67

 

2.41

B

2

1

0

0

1

1

 

2000.0

A

12

12

14

13

13

14

 

21.67

 

31.63

B

0

0

0

0

0

0

 

2500.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

3000.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

3300.0

A

 

n.c.²

 

n.c.²

 

n.c.²

B

 

DMBA1.25

A

48

55

58

40

29

41

 

169.44

 

310.70

B

55

69

57

60

48

50

 

a = number of colonies 7 days after seeding about 300 000 cells/flask in selection

medium

b = correction on the basis of the absolute cloning efficiency 2 at the end of the

expression period

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 cytotoxicit

Applicant's summary and conclusion

Conclusions:
In the presence of metabolic activation, Azo Initiator VA044 induces gene mutations in the HPRT locus assay using CHO cells under the experimental conditions chosen
Executive summary:

The substanceAzo Initiator VA044was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of liver S9 mix from 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 (4-hour exposure period)

0; 62.5;125.0; 250.0; 500.0; 1 000.0;2 000.0μg/mL

with S9 mix (4-hour exposure period)

0; 125.0; 250.0; 500.0; 1 000.0; 2 000.0;3 300.0μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 78.1; 156.3; 312.5; 625.0; 1 250.0;2 500.0μg/mL

with S9 mix (4-hour exposure period)

0;156.3; 312.5; 625.0; 1 250.0; 2 500.0; 3 300.0μg/mL

3rd Experiment

with S9 mix (4-hour exposure period)

0; 1 000.0; 1 500.0; 2 000.0;2 500.0; 3 000.0; 3 300.0μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and the 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 negative controls gave mutant frequencies within our historical negative control data range for CHO cells. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]anthracene (DMBA), led to the expected increase in the frequencies of forward mutations.

In in all experimental parts of this study at least the highest concentrations tested for gene mutations were clearly cytotoxic in the absence and presence of metabolic activation. Based on the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the mutant frequencies in the presence of metabolic activation in the 2nd and 3rd Experiment. These observations occurred at cytotoxic concentrations only.