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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:
Dec 2009 - Feb 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
July 2016
GLP compliance:
yes (incl. certificate)
Remarks:
Quality Assurance statement, 4 May 2010
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.: DEJ30990H0SOURCE OF TEST MATERIAL
- Analytical purity: >= 99.9 %
- Expiration date of the lot/batch: 30 Oct. 2011

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25°C
- Solubility and stability of the test substance in the solvent/vehicle: preliminary solubility tests revealed limit concentration of 906.8 µg/mL in culture medium, solubility in DMSO of at least 90.68 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: solution preperation under subdued lighting by formulating the test article in DMSOn(vortex mixing if required); The solutions were protected from light and used within 2 hours


Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology Incorporated, USA; Sprague Dawley rats induced with Aroclor 1254
- method of preparation of S9 mix : G6P (180 mg/mL), NADP (25 mg/mL), KCl (150mM) and S9 were mixed in ratio 1:1:1:2
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL of S9-mix to each cell culture (19 mL)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Sterility test, protein content analysis, test for presence of adventitious agents, promutagen activation
Test concentrations with justification for top dose:
Range finder: 22.86; 45.71; 91.43; 182.9; 365.7; 731.4 µg/mL (with and without S-9 mix)
Experiment 1: 100, 200, 300, 350, 400, 450, 500, 550, 650, 731.4 (without S-9 mix)
100, 200, 300, 400, 500, 550, 600, 650, 700, 731.4 (with S-9 mix)
Experiment 2: 50, 100, 200, 300, 350, 400, 450, 500, 600, 731.4 (without S-9 mix)
100, 200, 300, 400, 450, 500, 550, 600, 650, 731.4 (with S-9 mix)

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Remarks:
4-nitroquinoline 1-oxide: 0.10/0.15 µg/ml (without S-9 mix); Benzo[a]pyrene: 2.00/3.00 µg/ml (with S-9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 7 days

SELECTION AGENT (mutation assays): 6GT, 15 µg/ml

NUMBER OF REPLICATIONS: 2 (single cultures used for the positive control treatments)

Evaluation criteria:
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
There was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
The effects described above were reproducible.
Results that only partially satisfy the assessment criteria described above are considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 731.4 µg/mL, were tested in the absence and presence of S 9. Following the treatment incubation period, the highest two concentrations in the absence of S-9 and the highest three concentrations in the presence of S-9 (650 to 731.4 µg/mL in each case) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest two remaining concentrations in the absence of S-9 (500 and 550 µg/mL) and the highest remaining concentration in the presence of S 9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 450 µg/mL in the absence of S 9 and 550 µg/mL in the presence of S 9, which gave 10% and 7% RS, respectively. In the presence of S-9, no concentration gave 10 20% RS. Cultures treated at 500 and 550 µg/mL gave 21% and 7% RS, respectively, therefore both concentrations were analysed.

In Experiment 2, ten concentrations, ranging from 50 to 731.4 µg/mL in the absence of S-9 and from 100 to 731.4 µg/mL in the presence of S 9, were tested. Following the treatment incubation period, the highest two concentrations in the absence of S-9 (600 and 731.4 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S 9 (500 µg/mL) and the highest four concentrations in the presence of S 9 (550 to 731.4 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations in the absence and presence of S-9 were selected. However, the concentration of 300 µg/mL in the presence of S-9 was later rejected from analysis due to extreme heterogeneity for viability. Marked heterogeneity (also for viability) was observed at concentrations of 50 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, but these were included for comparative purposes. The highest concentrations analysed were 450 µg/mL in the absence of S 9 and 500 µg/mL in the presence of S 9, which gave 10% and 13% RS, respectively.

Any other information on results incl. tables

Tab. 1 Summary of mutation data experiment 1

Experiment 1 (3 hours treatment in the absence and presence of S-9 mix)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

4.80

 

0

 

100

3.57

 

100

 

72

3.25

NS

100

 

96

3.14

NS

200

 

74

4.91

NS

200

 

69

2.75

NS

300

 

67

2.03

NS

300

 

62

3.91

NS

350

 

43

5.24

NS

400

 

62

4.65

NS

400

 

29

7.07

NS

500

 

21

2.87

NS

450

 

10

6.85

NS

550

 

7

6.33

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

44

44.33

 

2

 

52

21.21

 

0.15

 

51

56.73

 

3

 

23

56.04

 

 

 

 

 

 

 

 

 

 

 

 

 

§: 6TG resistant mutants/106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

Tab. 2 Summary of mutation data experiment 2

Experiment 2 (3 hours treatment in the absence and presence of S-9 mix)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.59

 

0

 

100

2.49

 

50

$$

80

1.85

 

100

 

127

3.84

NS

100

 

71

2.38

NS

200

 

76

3.36

NS

200

 

68

3.50

NS

400

 

45

2.70

NS

300

 

48

2.28

NS

450

$$

30

2.96

 

350

 

27

4.48

NS

500

 

13

5.28

NS

400

 

18

3.32

NS

 

 

 

 

 

450

 

10

10.10

*

 

 

 

 

 

Linear trend

 

*

Linear trend

 

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

61

24.70

 

2

 

58

39.09

 

0.15

 

41

21.20

 

3

 

32

69.84

 

 

 

 

 

 

 

 

 

 

 

 

 

§: 6TG resistant mutants/ 106 viable cells 7 days after treatment

%RS: Percent relative survival adjusted by post treatment cell counts

NS: Not significant

* : Comparison of each treatment with control: Dunnett#s test (one-sided), significant at 5% level

*,**,*** : Test for linear trend: χ2 (one-sided), significant at 5%, 1% and 0.1% level respectively

$$ : Treatment has marked heterogeneity for viability but is included for comparative purposes

Applicant's summary and conclusion

Conclusions:
The test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under the tested conditions.
Executive summary:

The test substance was investigated for mutation at the hprt locus in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finding experiment followed by two independent experiments (with and without rat S9 metabolic activation). The test article was formulated in DMSO. In the range-finding study concentrations of 22.86 - 731.4 µg/mL were tested. The highest concentration to provide more than 10% survival was 365.7 µg/mL. In experiment 1 ten concentrations (100 - 731.4µg/mL) were tested in the absence and presence of metabolic activation. Seven days after treatment a concentration of 450 µg/mL without S9-mix and 550 µg/mL with S9-mix gave 10% and 7% relative survival, respectively. In experiment 2, concentrations of 50 - 731.4 µg/mL without S9 -mix and 100 - 731.4 µg/mL with S9 mix were tested. Relative survival of 10% and 13% were determined for 450 µg/mL with S9-mix and 500 µg/mL without S9-mix, respectively. Positive and negative control data indicated the study validity. Testing with concentrations up to toxicity (450 µg/mL) in the abscence of S9 in experiment 2 revealed a significant increase in mutant frequency. However, the result was not reproducable with experiment 1and according to the acceptance criteria not of biological relevance. In conclusion, the test substance did not induce mutation at the hprt locus under the test conditions employed in this study.