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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:
Sep 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
GLP compliance:
yes (incl. certificate)
Remarks:
GLP-Certificate (from the competent authority) Rheinland-Pfalz Landesamt für Umwelt
Type of assay:
other: Gene Mutation Assay in Mammalian Cells in vitro (HPRT)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Remarks:
slightly yellow
Details on test material:
- Molecular weight: 225.25 g/mol
- State of aggregation: solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE, Batch No. 0016908122
- Expiration date of the lot/batch: 2021-03-24
- Purity: 99.72 %
- State of aggregation: solid, slightly yellow

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until 24 Mar 2021 as indicated by the sponsor and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance at room temperature in the vehicle THF over a period of 4 hours was verified analytically.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The substance was dissolved in THF. The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly. The further concentrations were diluted 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
- Cell cycle length, doubling time or proliferation index: doubling time about 12 - 16 hours
- Methods for maintenance in cell culture if applicable: For cell cultivation, deep-frozen cell suspensions were thawed at 37 °C in a water bath, and volumes of 0.5 mL were transferred into 25 cm² plastic flasks containing about 5 mL Ham's F12 medium including 10 % (v/v) FCS. Cells were grown with 5 % (v/v) CO2 at 37 °C and >= 90 % relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- 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 supplemented with 10 % fetal calf serum (FCS), 1 % (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 µg/mL) and 1 % (v/v) amphotericine B (stock solution: 250 µg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat liver phenobarbital and ß-naphthoflavone induced S9 fraction)
Test concentrations with justification for top dose:
Test concentrations were 0, 3.13, 6.25, 12.50 and 25.00 µg/mL in the 1st experiment (with and without metabolic activation) and 0, 5.00, 10.00, 20.00 and 25.00 µg/mL in the 2nd experiment (with and without metabolic activation). The tested concentrations for the dose selection are listed in Tables 1 and 2.
The doses / concentrations tested in this study were selected in pre-tests. Precipitation of the test substance in the vehicle THF was not observed in the stock solution (test group: 500 µg/mL). In culture medium, test substance precipitation occured by the end of treatment at concentrations of 31.3 µg/mL and above in the absence of S9 mix and at concentrations of 3.9 µg/mL and above in the presence of S9 mix. After 4 hours treatment in the absence of S9 mix, no cytotoxicity was observed as indicated by a reduced Relative Survival of about or below 20 % of control. In contrast, in the presence of S9 mix, a clearly reduced relative Relative Survival was observed after treatment with 31.3 µg/mL and above.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetramethylhydrofuran (THF)
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, THF 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 THF in culture medium was 0.5 % (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
- Cell density at seeding (if applicable): about 20 x 10^6

DURATION
- Preincubation period: about 20 - 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): about 6 - 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

STAIN: Giemsa

NUMBER OF CELLS EVALUATED: 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.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95 % control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not "under control".
- The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range.

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 exceeds both the concurrent negative/vehicle control value and the range of our laboratory's historical negative control data (95 % control limit).

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 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 (propability 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 are printed in the tables.
However, both, biological and statistical significance are considered together.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
but tested up to precipitation
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: In this study, in the absence and presence of S9 mix, test substance was observed macroscopically in culture medium at the end of treatment at 25.00 µg/mL and above in the 1st and the 2nd experiment, respectively.

RANGE-FINDING/SCREENING STUDIES: In the 1st pretest DMSO was selected as vehicle. However, no homogenous solution in DMSO could be achieved. Thus, a 2nd pretest with THF as solvent was performed.
In the 2nd pretest for toxicity based on the solubility properties of the test substance 500.00 µg/mL (approx. 2.2 mM) test substance was used as top concentration both with and without S9 mix at 4 hour exposure time.
Higher concentrations could not be formulated in a suitable solvent.
The pretest was performed following the method described for the main experiment. The Relative Survival (RS) was determined as a toxicity indicator for dose selection.

Any other information on results incl. tables

Table 1: Pretest: 4 hour exposure; without S9 mix

Test groups

Doses

pH*

Change

Solubility

Relative Survival [%]

Veh

Ma

Culture medium

0 h

3 – 4 h

Ma

Ma

Mi

Vehicle control**

No

n.d.

n.d.

n.d.

n.d.

100.0

2.0 µg/mL

No

S

S

S

S

83.9

3.9 µg/mL

No

S

S

S

S

78.7

7.8 µg/mL

No

S

S

S

S

92.5

15.6 µg/mL

No

S

S

S

S

87.2

31.3 µg/mL

No

S

P

P

P

94.9

62.5 µg/mL

No

S

P

P

P

91.9

125.0 µg/mL

No

S

P

P

P

98.8

250.0 µg/mL

No

S

P

P

P

103.4

500.0 µg/mL

No

S

P

P

P

98.4

* = visual examination by pH indicator phenol red; normal range: about 6.7 - 8.3

** = THF 0.5 % (v/v)

n.d. = not determined

Ma = macroscopically

Mi = microscopically

S = solution

P = precipitation

Table 2: Pretest: 4 hour exposure; with S9 mix

Test groups

Doses

pH*

Change

Solubility

Relative Survival [%]

Veh

Ma

Culture medium

0 h

3 – 4 h

Ma

Ma

Mi

Vehicle control**

No

n.d.

n.d.

n.d.

n.d.

100.0

2.0 µg/mL

No

S

S

S

S

82.3

3.9 µg/mL

No

S

S

P

S

80.2

7.8 µg/mL

No

S

S

P

S

82.6

15.6 µg/mL

No

S

S

P

S

21.3

31.3 µg/mL

No

S

P

P

S

10.6

62.5 µg/mL

No

S

P

P

S

5.5

125.0 µg/mL

No

S

P

P

S

5.9

250.0 µg/mL

No

S

P

P

S

5.1

500.0 µg/mL

No

S

P

P

S

7.7

* = visual examination by pH indicator phenol red; normal range: about 6.7 - 8.3

** = THF 0.5 % (v/v)

n.d. = not determined

Ma = macroscopically

Mi = microscopically

S = solution

P = precipitation

Table 3: Mutant frequency and viability - 1st experiment without S9 mix; 4 -hour exposure period

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

uncorrected

Correctedb

Vehicle control*

137

145

70.5

100.0

1

7

2.00

2.84

1.56

n.c.1

n.c.1

3.13

131

134

66.3

94.0

1

2

0.75

1.13

6.25

120

151

67.8

96.1

2

1

0.75

1.11

12.50

106

120

56.5

80.1

4

7

2.75

4.87

25.00

110

111

55.3

78.4

11

3

3.50

6.33

50.00

n.c.²

n.c.²

100.00

EMS 400.0

118

107

56.3

79.8

203

186

97.25

172.89s

* = THF 0.5 % (v/v)

s = statistical significance (p =< 0.05)

a = number of colonies 7 days after seeding about 2x10^6 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 since only one concentration beyond the solubility limit is required

Table 4: Mutant frequency and viability - 1st experiment with S9 mix; 4 -hour exposure period

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

uncorrected

Correctedb

Vehicle control*

171

143

78.5

100.0

6

6

3.00

3.82

0.78

n.c.1

n.c.1

1.56

3.13

154

131

71.3

90.8

9

8

4.25

5.96

6.25

112

96

52.0

66.2

5

9

3.50

6.73

12.50

105

144

62.3

79.3

6

7

3.25

5.22

25.00

149

114

65.8

83.8

1

2

0.75

1.14

50.00

n.c.²

n.c.²

100.00

DMBA 1.25

99

96

48.8

62.1

239

217

114.0

233.85s

* = THF 0.5 % (v/v)

s = mutant frequency statistically significant higher than corresponding control values (p =< 0.05)

a = number of colonies 7 days after seeding about 2x10^6 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 since only one concentration beyond the solubility limit is required

Table 5: Mutant frequency and viability - 2nd experiment without S9 mix; 4 -hour exposure period

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

uncorrected

Correctedb

Vehicle control*

147

156

75.8

100.0

3

3

1.50

1.98

2.50

n.c.1

n.c.1

5.00

157

141

74.5

98.3

4

2

1.50

2.01

10.00

162

136

74.5

98.3

6

3

2.25

3.02

20.00

163

162

81.3

107.3

6

8

3.50

4.31

25.00

168

161

82.3

108.6

7

3

2.50

3.04

30.00

n.c.²

n.c.²

40.00

80.00

EMS 400.0

114

109

55.8

73.6

238

222

115.00

206.28s

* = THF 0.5 % (v/v)

s = mutant frequency statistically significant higher than corresponding control values (p =< 0.05)

a = number of colonies 7 days after seeding about 2x10^6 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 since only one concentration beyond the solubility limit is required

Table 6: Mutant frequency and viability - 2nd experiment without S9 mix; 4 -hour exposure period

Test groups [µg/mL]

Cloning efficiency 2 (viability)

Mutant frequency

Number of colonies

CE2

Number of coloniesa

MF (per 106cells)

Dish 1

Dish 2

Abs. [%]

Rel. [%]

Flask 1

Flask 2

uncorrected

Correctedb

Vehicle control*

161

159

80.0

100.0

1

0

0.25

0.31

2.50

n.c.1

n.c.1

5.00

180

141

80.3

100.3

7

7

3.50

4.36s

10.00

146

145

75.8

90.9

1

1

0.50

0.69

20.00

155

129

71.0

88.8

4

3

1.75

2.46

25.00

131

139

67.5

84.4

2

3

1.25

1.85

30.00

n.c.²

n.c.²

40.00

80.00

DMBA 1.25

103

106

52.3

65.3

149

122

67.75

129.67s

* = THF 0.5 % (v/v)

s = mutant frequency statistically significant higher than corresponding control values (p =< 0.05)

a = number of colonies 7 days after seeding about 2x10^6 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 since only one concentration beyond the solubility limit is required

Table 7: Cytotoxicity data - 1st experiment without S9 mix; 4 -hour exposure period

Test group [µg/mL]

Cell density at the end of treatment [x 105/mL]

Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)

Relative survival [%]

Number of colonies

Absolute [%]

Relative [%]

Adjusted [%]

Dish 1

Dish 2

Vehicle control*

7.53

156

188

86.0

100.0

129.5

100.0

1.56

8.17

162

170

83.0

96.5

135.6

104.7

3.13

8.00

194

187

95.3

110.8

152.4

117.7

6.25

7.07

167

156

80.8

93.9

114.2

88.2

12.50

8.40

171

145

79.0

91.9

132.7

102.5

25.00

6.54

126

170

74.0

86.0

96.8

74.7

50.00

6.21

174

154

82.0

95.3

101.8

78.6

100.00

7.15

165

163

82.0

95.3

117.3

90.5

EMS 400.00

9.45

129

115

61.0

70.9

115.3

89.0

* = THF 0.5 % (v/v)

Table 8: Cytotoxicity data - 1st experiment with S9 mix; 4 -hour exposure period

Test group [µg/mL]

Cell density at the end of treatment [x 105/mL]

Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)

Relative survival [%]

Number of colonies

Absolute [%]

Relative [%]

Adjusted [%]

Dish 1

Dish 2

Vehicle control*

2.80

168

165

83.3

100.0

46.6

100.0

0.78

3.01

174

173

86.8

104.2

52.2

112.0

1.56

3.41

144

161

76.3

91.6

52.0

111.5

3.13

3.35

189

173

90.5

108.7

60.6

130.1

6.25

2.82

190

188

94.5

113.5

53.3

114.3

12.50

2.99

147

194

85.3

102.4

51.0

109.4

25.00

3.60

149

151

75.0

90.1

54.0

115.8

50.00

6.73

95

71

41.5

49.8

55.9

119.8

100.00

7.15

84

82

41.5

49.8

59.3

127.3

DMBA 1.25

1.63

100

107

51.8

62.2

16.9

36.2

* = THF 0.5 % (v/v)

Table 9: Cytotoxicity data - 2nd experiment without S9 mix; 4 -hour exposure period

Test group [µg/mL]

Cell density at the end of treatment [x 105/mL]

Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)

Relative survival [%]

Number of colonies

Absolute [%]

Relative [%]

Adjusted [%]

Dish 1

Dish 2

Vehicle control*

6.54

165

179

86.0

100.0

112.5

100.0

2.50

8.64

188

200

97.0

112.8

167.6

149.0

5.00

8.53

203

191

98.5

114.5

168.0

149.4

10.00

9.43

201

186

96.8

112.5

182.5

162.2

20.00

9.58

193

154

86.8

100.9

166.2

147.8

25.00

9.51

187

203

97.5

113.4

185.4

164.9

30.00

9.50

n.c.²

40.00

10.08

80.00

9.25

EMS 400.00

11.20

166

170

84.0

97.7

188.2

167.3

* = THF 0.5 % (v/v)

n.c.2 = culture was not continued since only one concentration beyond the solubility limit is required

Table 10: Cytotoxicity data - 2nd experiment with S9 mix; 4 -hour exposure period

Test group [µg/mL]

Cell density at the end of treatment [x 105/mL]

Cloning efficiency 1 (survival) (4 h after treatment; 200 cells/dish seeded)

Relative survival [%]

Number of colonies

Absolute [%]

Relative [%]

Adjusted [%]

Dish 1

Dish 2

Vehicle control*

8.04

177

169

86.5

100.0

139.1

100.0

2.50

8.94

198

182

95.0

109.8

169.9

122.1

5.00

7.05

181

175

89.0

102.9

125.5

90.2

10.00

6.53

202

195

99.3

114.7

129.6

93.2

20.00

7.25

188

149

84.3

97.4

122.2

87.8

25.00

12.85

102

106

52.0

60.1

133.6

96.1

30.00

7.76

n.c.²

40.00

9.20

80.00

9.40

DMBA 1.25

5.97

124

155

69.8

80.6

83.3

59.9

* = THF 0.5 % (v/v)

n.c.2 = culture was not continued since only one concentration beyond the solubility limit is required

Applicant's summary and conclusion

Conclusions:
In the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
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 ß-naphthoflavone induced rats (exogenous metabolic activation).

According to two initial range-finding cytotoxicity tests for the determination of the experimental doses and based on the solubility properties of the test substance in culture medium the following concentrations were tested. Test groups printed in bold type were evaluated for mutant frequencies:

1st Experiment

without S9 mix

0; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

with S9 mix

0; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00; 100.00 µg/mL

2nd Experiment

without S9 mix

0; 2.50; 5.00; 10.00; 20.00; 25.00; 30.00; 40.00; 80.00 µg/mL

with S9 mix

0; 2.50; 5.00; 10.00; 20.00; 25.00; 30.00; 40.00; 80.00 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.

Based on the solubility properties of the test substance in culture medium 100.0 µg/mL was the highest applied concentration for genotoxicity testing. In both experiments, test substance precipitation occured in the highest applied concentrations (25.00 µg/mL onward) in culture medium 4 hours at the end of exposure period.

In both experiments in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations.

In the 1st experiment in the absence of a metabolizing system a dose related increase in the mutant frequencies of cultures treated was observed. The highest evaluated value of the test group 25.00 µg/mL (MFcorr.: 6.33 per 10^6 cells) exceeded the upper limit of the 95 % level of the historical control values (MFcorr.: 5.97 per 10^6 cells). However, the value obtained at 25.00 µg/mL was not statistically significant as compared to the corresponding control value and was within the range of our historical negative control data (MFcorr.: 0.00 - 6.48 per 10^6 cells). In the 2nd experiment in the absence of S9 mix, the observed concentration related increase as well as the observed increase in the mutation frequency at 25.00 µg/mL was not observed. Thus, the increase observed in the 1st experiment at 25.00 µg/mL is considered as biologically irrelevant.

In the presence of a metabolizing system the corrected mutation frequencies ranged between 1.14 - 6.73 per 10^6 cells and 0.69 - 4.36 per 10^6 cells in the 1st and 2nd experiment, respectively. The vehicle control values were (corrected mutation frequency: 3.82 and 0.31 per 10^6 cells) in the 1st and 2nd experiment, respectively. In the 2nd experiment, the corrected mutation frequency obtained at 5.00 µg/mL (4.36 mutants per 10^6 cells) was statistically significant. However, this observation is not biologically relevant, since it is neither concentration related nor exceeds the 95 % level of the historical control data.

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 the in vitro conditions in CHO cells in the absence and the presence of metabolic activation.